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SCIENCE REPEAL hc^l^o' iWfQ^m aTIQN SCR VICE

WORLD INTELLECTUAL PROPERTY ORGANIZATION ,
International Bureau

PCT

. INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification & : ■

C12N 15/12, C07K 14/705, 16/28, C12N
5/20

(11) International Publication Number: [ WO 97/22698

(43) International Publication Date: 26 June 1997 (26.06.97)

(21) International Application Number: PCT/US96/20759

(22) International Filing Date: 20 December 1996 (20.12.96)

(30) Priority Data:

08/575,967
08/661,3^3

20 December 1995 (20.12.95) US
7 June 1996 (07.06.96) US

(71) Applicant: ICO S CORPORATION [US/US]; 22021 20th

Avenue S.E., Bothell, WA 98021 (US).

(72) Inventors: GRAY, Patrick, W.; 1600 40th Avenue, Seattle,

WA 98122 (US). SCHWEICKART, Vicki, L.; 1421 Orange
Place North, Seattle, WA 98109 (US). RAPORT, Carol, J.;
2300 21 1th Street, S.E.. Bothell, WA 98021 (US).

(74) Agent: BORUN, Michael, F.; Marshall, O'Toole, Gerstein,
Murray & Borun, 6300 Sears Tower, 233 South Wacker
Drive, Chicago, IL 60606-6402 (US).

(81) Designated States: AU, BR, CA, CN, CZ, FI, HU, JP, MX,
NO, PL, RU, SK, European patent (AT, BE, CH, DE, DK,
ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).

Published

Without international search report and to be republished
upon receipt of that report.

(54) Title: CHEMOKINE RECEPTORS 88-2B[CKR-3] AND 88C AND THEIR ANTIBODIES
(57) Abstract

The present invention provides polynucleotides that encode the chemokine receptors 88-2B or 88C and materials and methods for
the recombinant production of these two chemokine receptors. Also provided are assays utilizing the polynucleotides which facilitate the
identification of ligands and modulators of the chemokine receptors. Receptor fragments, ligands, modulators, and antibodies are useful in
the detection and treatment of disease states associated with the chemokine receptors such as atherosclerosis, rheumatoid arthritis, tumor
growth suppression, asthma, viral infection, AIDS, and other inflammatory conditions.

■ - . , •. • i

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Armenia

United Kingdom

Malawi

Austria

Georgia

Mexico

Australia

Guinea

NE -

Niger

Barbados

Greece

Netherlands

Belgium

Hungary

Norway

Burkina Faso

Ireland

New Zealand

Bulgaria

Italy

Poland

Benin -

Japan

Portugal

Brazil

Kenya

Romania

Belarus

Kyrgystan

Russian Federation

Canada

Democratic People's Republic

Sudan

Central African Republic

of Korea

Sweden

Congo

Republic of Korea

Singapore

Switzerland

Kazakhstan

Slovenia

C6te d'lvohx

Liechtenstein -

Slovakia

Cameroon

Sri Lanka

Senegal

China

Liberia

. Swaziland

Czechoslovakia

Lithuania

.,. Chad

Czech Republic

Luxembourg

Togo

Germany

Latvia

Tajikistan

Denmark

Monaco

Trinidad and Tobago

Estonia

Republic of Moldova

Ukraine

Spain

Madagascar

Uganda

Finland

Mali

United States of America

France

Mongolia

Uzbekistan

Gabon

Mauritania

Viet Nam

WO 97/22698 PCT/US96/20759

CHEMOKINE RECEPTORS 88-2B [CKR-3] AND 88C AND THEIR ANTIBODIES

This application is a continuation-in-part of U.S. Patent
Application , Serial No. 08/661,393 filed June 7, 1996 which was in turn a
5 continuation-in-part of U.S. Patent Application No. 08/575,967 filed
December 20, 1995.

FIELD OF THE INVENTION

. The present invention relates generally to signal transduction
pathways. More particularly, the present invention relates to chemokine
10 receptors, nucleic acids, encoding chemokine receptors, chemokine receptor
ligands. modulators of chemokine receptor activity, antibodies recognizing
chemokines and chemokine receptors, methods for identifying chemokine
receptor ligands and modulators, methods for producing chemokine receptors, -
and methods for producing antibodies recognizing chemokine receptors.

15 BACKGROUND OF THE INVENTION

Recent advances in molecular biology have led to an
appreciation of the central role of signal transduction pathways in biological
processes. These pathways comprise a central means by which individual
cells in a multicellular organism communicate, thereby coordinating biological

20 processes. See Springer, Cell 7(5:301-314 (1994), Table I for a model. One
branch of signal transduction pathways, defined by the intracellular
participation of guanine nucleotide binding proteins (G-proteins) , affects a
broad range of biological processes. ' - «

Lewin, GENES V 3 19-348 (1994) generally discusses G-protein

25 signal transduction pathways which involve, at a minimum, the following
components: an extracellular signal (e.g., neurotransmitters, peptide
hormones, organic molecules, light, or odorants), a signal-recognizing
receptor (G-protein-coupled receptor, reviewed in Probst et al. , DNA and Cell
Biology 11: 1-20 [1992] and also known as GPR or GPCR), and an intracellu-

30 lar. heterotrimeric GTP-binding protein, or G protein. In particular, these

WO 97/22698 PCTYUS96/20759

. . ' ■ : - 2 -

pathways have attracted interest because of their role in regulating white blood
eel] or leukocyte trafficking.

Leukocytes comprise a group of mobile blood cell types
including granulocytes- '(i.e., neutrophils, basophils; and eosinophils),
5 lymphocytes, and monocytes. When mobilized and activated, these cells are
primarily involved in the body's defense against foreign matter. This task is
complicated by the diversity of normal and pathological processes in which
leukocytes participate. For example, leukocytes function in the normal
inflammatory response to infection. Leukocytes are also involved in a variety

10 - of pathological inflammations. For a summary, see Schall et al. , Curr. Opin.
Immunol. 6:865-873 (1994). Moreover, each of these processes can involve
unique contributions, in degree, kind, and duration, from each. of. the
leukocyte cell types.

In studying these immune reactions, researchers initially

15 concentrated on the signals acting upon leukocytes, reasoning that a signal
would be required to elicit any form of response. Murphy, Ann. Rev.
Immunol. 72:593-633 (1994) has reviewed members of an important group of
leukocyte signals, the peptide signals. One type of peptide signal comprises
the chemokines (c/zemoattractant cytokines), termed intercrines in Oppenheim

20 et oL 9 Ann. Rev. Immunol. .9:617-648 (1991). In addition to Oppenheim et
aL . Baggiolini et al. , Advances in Immunol. 55:97-179 (1994), documents the
growing number of chemokines that have been identified and subjected to
genetic and biochemical analyses.

Comparisons of the amino acid sequences of the known

25 chemokines have led to a classification scheme which divides chemokines into
two groups: the or group characterized by a single amino acid separating the
first two cysteines (CXC: N-tenninus as referent), and the (3 group, where
these cysteines are adjacent (CC). See Baggiolini et al., supra. Correlations
have been found between the chemokines and the particular leukocyte cell

30 types responding to those signals. Scltall et aL, supra, has reported that the

CXC chemokines generally affect neutrophils; the CC chemokines tend to .
affect monocytes,

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lymphocytes, basophils and eosinophils. For example, Baggiolini et aL r
supra, recited that RANTES, a CC chemokine, functions as a chemoattractant
for monocytes, lymphocytes (i.e., memory T cells), basophils, and
eosinophils, but not for neutrophils, while inducing the release of histamine

5 . from basophils.

Chemokines were recently shown by Cocchi, et. aL, Science,
270: 1811-1815 (1995) to be suppressors Of HIV proliferation. Cocchi, et aL
demonstrated that RANTES, MlP-la, and MIP-1/3 suppressed HIV-1 , HIV-2
and SIV infection of a CD4 + cell line designated PM1 and of primary human

10 peripheral blood mononuclear cells.

Recently , however, attention has turned to the cellular receptors
that bind the chemokines, because the extracellular chemokines seem to
contact cells indiscriminately, and therefore lack the specificity needed to
regulate the individual leukocyte cell types. ^

15 Murphy, supra, reported that the GPCR superfamily of

receptors includes the chemokine receptor family. The typical chemokine
receptor structure includes an extracellular chemokine-binding domain located
near the N-terminus, followed by seven spaced regions of predominantly
hydrophobic amino acids capable of forming membrane-spanning a-helices.

20 Between each of the a-helical domains are hydrophilic domains localized,
alternately, in the intra- or extra-cellular spaces. These features impart a
serpentine conformation to the membrane-embedded chemokine. receptor. The
third intracellular loop typically interacts with G-proteins. In addition,
Murphy, supra, noted that the intracellular carboxyl terminus is also capable

25 . of interacting with G-proteins.

The first chemokine receptors to be analyzed by molecular
cloning techniques were the two neutrophil receptors for human IL8, a CXC
chemokine. Holmes et aL , Science 253: 178-1280 (1991) and Murphy et al. ,
Science 253: 1280-1283 (1991) , reported the cloning of these two receptors for .

30 IL8. Lee et aL, J. Biol. Chan. 267: 16283- 16287 (1992), analyzed the
cDNAs encoding these receptors and found 77% amino acid identity between
the encoded receptors, with each receptor exhibiting features of the G protein

WO 97/22698 PCT7US96/20759

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coupled receptor family. One of these receptors is specific for IL-8, while the
other binds and signals in response to IL-8, gro/MGSA, and NAP-2. Genetic
manipulation of the genes encoding IL-8 receptors has contributed to our
understanding of the biological roles occupied by these receptors. For
5 example. Cacalano.et aL, Science 265:682-684 (1994) reported that deletion
of the IL-8 receptor homolog in the mouse resulted in a pleiotropic phenotype
involving lymphadenopathy and splenomegaly. In addition, a study of
missense mutations described in Leong et aL, J. Biol. Chem. 269: 19343-
19348 (1994) revealed amino acids in the IL-8 receptor that were critical for
10 IL-8 binding. Domain swapping experiments discussed ' in Murphy, supra,
implicated the amino terminal extracellular domain as a determinant of binding
specificity.

Several receptors for CC chemokines have also been identified
and cloned. CCCKR1 binds both MIP-la and RANTES and causes -
15 intracellular calcium ion flux in response to both ligands. Charo et aL, Proc
NarL Acad. Sci. (USA) P7:2752-2756 (1994) reported that another CC
chemokine receptor, MCP-R1 (CCCKR2), is encoded by a single gene that
produces two splice variants which differ in their carboxyl terminal domains.
This receptor binds and responds to MCP-3 in addition to MCP-1.

20 A promiscuous receptor that binds both CXC and CC

chemokines has also been identified. This receptor was originally identified
on red blood cells and Horuk et aL, Science 261: 11 82-1 184.. (1993) reports
that it binds IL-8, NAP-2, GROor, RANTES, and MCP-1. The erythrocyte
chemokine receptor shares about 25 % identity with other chemokine receptors

25 and may help to regulate circulating levels of chemokines or aid in the
presentation of chemokines to their targets. In addition to binding
chemokines, the erythrocyte chemokine receptor has also been shown to be the
receptor for Plasmodium vivax, a major cause of malaria (id.) Another G-
protein coupled receptor which is closely related to chemokine receptors, the

30 platelet activating factor receptor, has also been shown* to be the receptor for
a human pathogen, the bacterium Streptococcus pneumoniae (Cundell et aL,
Narure 377:435.-438 (1995)).

WO 97/22698 PCT/US 96/20759

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In addition to the mammalian chemokine receptors, two viral
chemokine receptor homologs have been identified. Ahuja et aL , J. BioL
Chem. 265:20691-20694 (1993) describes a gene product from Herpesvirus
saimiri that shares about 30% identity with the IL-8 receptors and binds CXC
5 chemokines. Neote et aL, Cell, 72/415-425 (1993) reports that human
cytomegalovirus contains a gene encoding a receptor , sharing about 30%
identity with the CC chemokine receptors which binds MIP-lcx. MIP-1/?,
MCP-1, and RANTES. These viral receptors may affect the normal role of
chemokines and provide a selective pathological advantage for the virus.

10 . Because of the broad diversity of chemokines and their

activities; there are numerous receptors for the chemokines. The receptors
which : have been characterized represent only a fraction of the total
complement of chemokine receptors. There, thus remains a need in the art for
the identification of additional chemokine receptors. The availability of these

15 novel receptors will provide tools for the . development of therapeutic
modulators of chemokine or chemokine receptor function. It is contemplated
by the present invention that such modulators are useful as therapeutics for the
treatment of atherosclerosis, rheumatoid arthritis, tumor growth suppression,
asthma, viral infections, and other inflammatory conditions. Alternatively,

20 fragments or variants of the chemokine receptors, or antibodies recognizing
, those receptors, are contemplated as therapeutics.

SUMMARY OF THE INVENTION

The present invention provides purified and isolated nucleic
acids encoding chemokine receptors involved in leukocyte trafficking.

25 Polynucleotides of the invention (both sense and anti-sense strands thereof)
include genomic DNAs, cDNAs, and RNAs, as well as completely or partially
synthetic nucleic acids. Preferred polynucleotides of the invention include the
DNA encoding the chemokine receptor 88-2B that is set out in SEQ ID NO: 3,
the DNA encoding the chemokine receptor 88C that is set out in SEQ ID

30 NO:l, and DNAs which hybridize to those DNAs under standard stringent
hybridization conditions, or which would hybridize but for the redundancy of

• WO 97/22698 PCT/US96/20759

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the genetic code. Exemplary stringent hybridization conditions are as follows:
hybridization at 42°C in 50% formamide, 5X SSC, 20 mM sodium phosphate,
pH 6.8 and washing in 0.2X SSC at 55 J C. It is understood by those of skill
in the art that variation in these conditions occurs based on the length and GC
5 nucleotide content of the sequences to be hybridized. Formulas standard in
the art are appropriate for determining exact hybridization conditions. See
Sambrook et aL, §§ 9.47-9.51 .in Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
. (1989). Also contemplated by the invention are polynucleotides encoding *

1 0 domains of 88-2B or 88C, for example, polynucleotides encoding one or more
extracellular domains of either protein or other biologically active fragments
thereof- 88-2B extracellular domains correspond to SEQ ID NO: 3 and SEQ
ID NO: 4 at amino acid residues 1-36, 93-107, 171-196, and 263-284. The
extracellular domains ' of 88-2B are encoded by polynucleotide sequences

15 corresponding to SEQ ID NO:3 at nucleotides 362-469, 638-682, 872-949,
and 1148-1213. Extracellular domains of 88C correspond to SEQ ID.NO:l
and SEQ ID NO:2 at amino acid residues 1-3.2, 89-112, 166-191, and 259-
280. The 88C extracellular domains are encoded by polynucleotide sequences
that correspond to SEQ ID NO:l at nucleotides 55-150, 319-390, 550-627,

20 and 829-894. The invention also comprehends polynucleotides encoding
intracellular domains of these chemokine receptors. The intracellular domains
of 88-2B include amino acids 60-71, 131-151, 219-240, and 306-355 of SEQ
ID NO:3 and SEQ ID NO:4. Those domains are encoded by polynucleotide
sequences corresponding to SEQ ID NO: 3 at nucleotides 539-574, 752-814,

25 1016-1081, and 1277-1426, respectively. The 88C intracellular domains
include amino acid residues 56-67, 125-145, 213-235, and 301-352 of SEQ
ID NO: 1 and SEQ ID NO:2. The intracellular domains of 88C are encoded
by polynucleotide sequences corresponding to SEQ ID NO: 1 at nucleotides
220-255, 427-489, 691-759, and 955-1 1 10. Peptides corresponding to one or

30 more of the extracellular or intracellular domains, or antibodies raised against
those peptides, are contemplated as modulators of receptor activities,
especially ligand and G protein binding activities of the receptors.

WO 97/22698

PCT/US 96/20759

- 7

The nucleotide sequences of the invention may also be used to
-«n oli g onucl, 0 tides for use as labeled probes „
encodin g 88-2B or SBC under stri n g ent hybridan CO ndLns
S^thern analyses and Polymerase Chain Reaction. .nethodo.ogies). Moreover
these ol lg on U cleotide probes can be use, to detect particular alleles of the

agents of dzsease states associated with particular allele, in addition
, e e oh g onucleotides can be used to alter chemolcine receptor g enetics to
n .dentification of chemolcine receptor modulator, Mso \ e
> nucleo.de sequences can be used to desi gn antisense g enetic events of £
. » explonng or altering the genetics and expression of 88-2B or 88C The

invention also comprehends bioloeicaJ mnli rac r ■ '

• oioiogicai replicas (i.e. ; copies of isolated DNA?

If " " W " * ^ ^ ™ A of ™As of tne invention

A U .o_, y rep,ica,i„ g recombfaant constmaions £uch as - ■

2 ; rrr r*- YAC) nuc,eic acid ^ ^ i-.^

88 2B or 88C po, y „uc,eotic,es, K particuIar)y> ^ wherein b J -
efl*c,,ve, y encoding 88 . 2B or 88C , opera(ive)y ljnked on J*

endogenous or „e,ero,o g ous egression contro , are ^

The 88-2B and 88C receptors may be produced naturally

-n.fonned or Greeted witn ^nucleotides o f t„e .nvention Dy J ^
~ be used K express ^ 8g ^ cheraokjne ^

o 8 ^ 8 T ^ — y active ^ent

or 55 m or 88C, analogs of 88-2B or RZr

fromth Pam - ' 8 ZB ° r 88C ' and Athene peptides derived

rrom the ammo acid sequences nfssm . . •

quences or 88-2B, set out m SEQ ID NO-4 or RRr

88 2B or 88C g ene product, or a biologically active f ragment of ^

product, when produced in a eukaryotic cell m . k
mnrIif . , . "Karyotic cell, may be post-translationailv

modified (e. p via disulfin^ . ^auy

Kg., d.sulfide bond fonnanon, glycosylation, phosphorylation
mynstoylation, palmitoylation, acetylation etc ) Th.
~ rt „, , ' eic -J Tn e invention further

contemplates the 88-2B and RSr 00 - ru ™er

and 88C gene productS) or 5ioIogicaUy ^

WO 97/22698 PCT/US96/20759

• - 8 - . .

fragments thereof, in monbmeric. homomultimeric, or . heteromultirneric
conformations.

In particular, one aspect of the invention involves antibody
products capable of specifically "binding to the 88-2B or 88C chemokine
5 receptors. The antibody products are generated by methods standard in the
an using recombinant 88-2B or 88C receptors, synthetic peptides or peptide
fragments of 88-2B or 88C receptors, host cells expressing 88-2B or 88C on
their surfaces, or 88-2B or 88C receptors purified from natural sources as
immunogens. The antibody products may include monoclonal antibodies or
10 polyclonal antibodies of any source or sub-type. Moreover, monomelic,
homomultimeric. and heteromultirneric antibodies, and fragments thereof, are
contemplated by the invention. Further, the invention comprehends CDR-
* grafted antibodies, "humanized" antibodies, and other modified antibody
products retaining the ability to specifically bind a chemokine receptor.
15 The invention also contemplates the use of antibody products

for detection of the 88--2B or 88C gene products, their analogs, or biologically
active fragments thereof. For example, antibody products may be used in
diagnostic procedures designed to reveal correlations between the expression
of 88-2B, or 88C, and various normal or pathological states. In addition,
20 antibody products can be used to diagnose tissue-specific variations in
expression of 88-2B or 88C, their analogs, or biologically active fragments
thereof. Antibody products specific for the 88-2B and 88C chemokine
receptors may also act as modulators of receptor activities. In another aspect,
antibodies to 88-2B or 88C receptors are useful for therapeutic purposes.
25 Assays for ligands capable of interacting with the chemokine

receptors of the invention are also provided. These assays may involve direct
detection of chemokine receptor activity, for example,- by monitoring the
binding of a labeled ligand to the receptor. In addition, these assays may be
used to indirectly assess ligand interaction with the chemokine receptor. As
30 used herein the term "ligand" comprises molecules which are agonists and
antagonists of 88-2B or 88C, and other molecules which bind to the receptors.

WO 97/22698 PCT/US96/20759

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Direct detection of ligand binding to a chemokine receptor miay
be achieved using the following assay. Test compounds {i.e., putative
ligands) are detectably labeled {e.g., radioiodinated). The detectably labeled
test compounds are then contacted with membrane preparations containing a
5 chemokine receptor of the invention. Preferably, the membranes are prepared
from host cells expressing chemokine receptors of the invention from
recombinant vectors. Following an incubation period to facilitate contact
between the membrane-embedded chemokine receptors and the detectably
labeled test compounds, the membrane material is collected on filters using
10 vacuum filtration. The detectable label associated with the filters is then
quantitated. For example, radiolabels are quantitated using liquid scintillation
spectrophotometry. Using this technique, ligands binding to chemokine
receptors are identified. To confirm the identification of a ligand, a detectably
labeled test compound is exposed to a membrane preparation displaying a
15 chemokine receptor in the presence of increasing quantities of the test
compound in an unlabeled state. A progressive reduction in the level of filter-
associated label as one adds increasing quantities of unlabeled test compound r
confirms the identification of that ligand.

Agonists are ligands which bind to the receptor and elicit
20 intracellular signal transduction and antagonists are ligands which bind to the
receptor but do not elicit intracellular signal transduction. The determination
of whether a particular ligand is an agonist or an antagonist can be
determined, for example, by assaying G protein-coupled signal transduction
pathways. Activation of these pathways can be determined by measuring
25 intracellular ca ++ flux, phospholipase C activity or adenylyl cyclase activity,
in addition to other assays (see examples 5 and 6).

As discussed in detail in the Examples herein, chemokines that
bind to the 88C receptor include RANTES, MIP-lo:, and MTP-10, and *
chemokines that bind to the 88-2B receptor include RANTES.
30 In another aspect, modulators of the interaction between the

88C and 88-2B receptors and their ligands are specifically contemplated by the
invention. Modulators of chemokine receptor function may be identified using

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assays similar to those used for identifying ligands. The membrane
preparation displaying a chemokine receptor is exposed to a constant and
known quantity of a detectably labeled functional ligand. " In addition, the
membrane-bound chemokine receptor is also exposed to an increasing quantity
5 of a test compound suspected of modulating the activity of that chemokine
receptor. If the levels of filter-associated label correlate with the quantity of
test compound, that compound is a modulator of the activity of the chemokine
receptor. If the level of filter-associated label increases with increasing
quantities of the test compound, an activator has been identified. In contrast,

10 if the level of filter-associated label varies inversely with the quantity of test
compound, an inhibitor of chemokine receptor activity has been identified.
Testing for modulators of receptor binding in this way allows for the rapid
screening of many putative modulators, as pools containing many potential
modulators can be tested simultaneously in the same reaction.

15 The indirect assays for receptor binding involve measurements

of the concentration or level of activity of any of the components found in the
relevant signal transduction pathway. Chemokine receptor activation often is
associated with an intracellular Ca ++ flux. Cells expressing chemokine.
receptors may be loaded with a calcium-sensitive dye. Upon activation of the

20 expressed receptor, a Ca ++ flux would be rendered spectrophotometrically
detectable by the dye. Alternatively, the Ca ++ flux could be detected
microscopically. Parallel assays, using either technique, may be performed
in the presence and absence of putative ligands. For example, using the
microscopic assay for Ca ++ flux, RANTES, a CC chemokine, was identified

25 as a ligand of the 88-2B chemokine receptor. Those skilled in the art will
recognize that these assays are also useful for identifying and monitoring the
purification of modulators of receptor activity. Receptor' activators and
inhibitors will activate or inhibit, respectively, the interaction of the receptors
with their ligands in these assays.

30 Alternatively,, the association of chemokine receptors with G

proteins affords the opportunity of assessing receptor activity by monitoring

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G protein activities. A characteristic activity of G proteins. GTP Hydrolysis,
may be monitored using, for example, 32 P-labeled GTP.

G proteins also affect a variety of other molecules through their
participation in signal transduction pathways. For example, G protein effector
5 molecules include adenylyl cyclase, phospholipase C, ion channels, and
phosphodiesterases. Assays focused on any of these effectors may be used to
monitor chemokine receptor activity induced by ligand binding in a host cell
that is both expressing the chemokine receptor of interest and contacted with
an appropriate ligand. For example, one- method by which the activity of

10 chemokine receptors may be detected involves measuring phospholipase C
activity. In this assay, the production of radiolabeled inositol phosphates by
host cells expressing a chemokine receptor in the presence of an agonist is
detected.. The detection of phospholipase activity may require cotransfection
with DNA encoding an exogenous G protein. When cotransfection is

15 required, this assay" can be performed by cotransfection of chimeric G protein
DNA, for example, Gqi5 (Conklin, et aL, Nature 553:274-276 (1993), with
88-2B or 88C DNA and detecting phosphoinositol production when the
cotransfected cell is exposed to an agonist of the 88-2B or 88C receptor.
Those skilled in the art will recognize that assays focused on G-protein

20 effector molecules are also useful for identifying and monitoring the
■ purification of modulators of receptor activity. Receptor activators and
inhibitors will activate or inhibit, respectively, the interaction of the receptors
* with their ligands in these assays.

Chemokines have been linked to many inflammatory diseases,

25 such as psoriasis, arthritis, pulmonary fibrosis and atherosclerosis. See
Baggiolini et aL, supra. Inhibitors of chemokine action may be useful in
treating these conditions. In one example, Broaddus et aL, J. of Immunol
752/2960-2967 (1994), describes an antibody to IL-8 which can inhibit
neutrophil recruitment in endotoxin-induced pleurisy, a model of acute.

30 inflammation in rabbit lung. It is also contemplated that ligand or modulator
* binding to, or the activation of, the 88C receptor may be useful in treatment
of HIV infection and HIV related disease states.. Modulators of chemokine

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binding to specific receptors contemplated by the invention may include
antibodies directed toward a chemokine or a receptor, biological or chemical
small molecules, or synthetic peptides corresponding to fragments of the
chemokine or receptor. .
5 Administration of compositions containing 88-2B or 88C

modulators to mammalian subjects, for the purpose of monitoring or
remediating normal or pathological immune reactions And viral infections
including infection by retroviruses such as HIV-1, HIV-2 and SIV is
contemplated by the invention. In particular, the invention comprehends the
10 mitigation of inflammatory responses, abnormal hematopoietic processes, and
viral infections by delivery of a pharmaceutically acceptable quantity of 88-2B
or 88C chemokine receptor modulators. The invention further comprehends
delivery of these active substances in pharmaceutically ; acceptable
compositions comprising carriers, diluents/ or medicaments. The invention
15 also contemplates a variety of administration routes. For example, the active
substances may be administered by the following routes: intravenous,
subcutaneous, intraperitoneal, intramuscular, oral, anal (i.e., via suppository
formulations), or pulmonary (i.e., via inhalers, atomizers, nebulizers, etc.)

In another aspect, the DNA sequence information provided by
20 the present invention makes possible the development, by homologous
recombination or "knockout" strategies [see, e.g. Kapecchi. Science,
244/1288-1292 (1989)], of rodents that fail to express a functional 88C or 88- .
2B chemokine receptor or that express a variant of the receptor.
Alternatively, transgenic mice which express a cloned 88-2B or 88C receptor
25 can be prepared by well known laboratory techniques (Manipulating the
Mouse Embryo: A Laboratory Manual, Brigid Hohan, Frank Costantini and
Elizabeth Lacy, eds. (1986) Cold Spring Harbor Laboratory ISBN 0-87969-
175-1). Such rodents are useful as models for studying the activities of 88C
or 88-2B receptors in vivo.
30 Other aspects and advantages of the present invention will

become apparent to one skilled in the art upon consideration of the following
examples.

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DETAILED DESCRIPTION OF THE INVENTION

The following examples illustrate the invention. Example 1
describes the isolation of genomic DNAs encoding the 88-2B and 88C
chemokine receptors. Example 2 presents the isolation and sequencing of •

5 cDNAs encoding human 88-2B and 88C and macaque 88C. Example 3
provides a description of Northern analyses revealing the expression patterns
of the 88-2B and 88C receptors in a variety of tissues. Example 4 details the
recombinant expression of the 88-2B and 88C receptors. Example 5 describes
Ca ++ flux assays, phosphoinositol hydrolysis assays, and binding assays for

10 88-2B and 88C receptor activity in response to a variety of potential ligands.
Experiments describing the role of 88C and 882B as co-receptors for HIV is
presented in Examples 6 and 7. The preparation and characterization of
monoclonal and polyclonal antibodies immunoreactive with 88C is described
in Example 8. Example 9 describes additional assays designed to identify 88-

15 2B or 88C ligands or modulators.

Example 1

Partial genomic clones encoding the novel chemokine receptor
genes, of this invention were isolated by PCR based on conserved sequences
found in previously identified genes and based on a clustering of these

20 chemokine receptor genes within the human genome. The genomic DNA was
amplified by standard PCR methods using degenerate oligonucleotide primers.

Templates for PCR amplifications were members of a
commercially available source of recombinant human genomic DNA cloned
into Yeast Artificial Chromosomes (i.e., YACs). (Research Genetics, Inc.,

25 Huntsville, AL, YAC Library Pools, catalog ho. 95011 B). A YAC vector
can accommodate inserts of 500-1000 kilobase pairs. Initially, pools of YAC
clone DNAs were screened by PCR using primers specific for the gene
encoding CCCKR1. In particular, CCCKR(2)-5', the sense strand primer
(corresponding to the sense strand of .CCCKRl), is presented in SEQ ID

30 NO: 15. Primer CCCKR(2)-5' consisted of the sequence 5'-
CGTAAGCTTAGAGAAGCCGGGATGGGAA-3' , wherein the underlined

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nucleotides are the translation start codon for CCCKR1 . The anti-sense strand
primer was CCCKR-3' (corresponding to the anti-sense strand of CCCKR1)
and its sequencers presented in SEQ ID NO: 16. The sequence of CCCKR-
3 ' , 5 ' -GCCTCTAGAGTC AG AG ACC AGC AG A-3 ' , contains the reverse

5 complement of the CCCKR1 translation stop codon (underlined). Pools of
YAC clone DNAs yielding detectable PCR products (i.e., DNA bands upon
gel electrophoresis) identified appropriate sub-pools of YAC clones, based on
a proprietary identification scheme. (Research Genetics, Inc., Huntsville,
AL). PCR reactions were initiated with an incubation at 94°C for four

10 minutes. Sequence amplifications were achieved using 33 cycles of
denaturation at 94"C for one minute, annealing at 55"C for one minute, and
extension at 72"C for two minutes.

The sub-pools of YAC clone DNAs were then subjected to a
second round of PCR reactions using the conditions, and primers, that were

15 used in the first round of PCR. Results from sub-pool screenings identified
individual clones capable of supporting PCR reactions with the CCCKR-
specific primers. One clone, 881F10, contained 640 kb of human genomic
DNA from chromosome * 3p21 including the genes for CCCKR1 and
CCCKR2, as determined by PCR and hybridization. An overlapping YAC

20 clone, 941A7, contained 700 kb of human genomic DNA and also contained
the genes for CCCKR1 and CCCKR2. Consequently, further mapping studies
were undertaken using these two YAC clones. Southern analyses revealed
that CCCKR1 and CCCKR2 were located within approximately 100 kb of one
another.

25 The close proximity of the CCCKR1 and CCCKR2 genes

suggested that novel related genes might be linked to CCCKR1 and CCCKR2.
Using DNA from yeast containing YAC clones 881F10 and 941A7 as
templates, PCR reactions were performed to amplify any linked receptor
genes. Degenerate oligodeoxy ribonucleotides were designed as PCR primers.

30 These oligonucleotides corresponded to regions encoding the second
intracellular loop and the sixth transmembrane domain of CC chemokine
receptors, as deduced from aligned sequence comparisons of CCCKR1,

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. ; - 15 -

CCCKR2, and V28. V28 was used because it is an orphan receptor that
exhibits the characteristics of a chemokine receptor; V28 has also been
mapped to human chromosome 3. Raporr ex al, Gene 163:295-299 (1995).
Of further 'note, the two splice variants of CCCKR2. CCCKR2A and
5 CCCKR2B, are identical in the second intracellular loop and sixth
transmembrane domain regions used in the analysis. . The 5' primer,
designated V28degf2, contains an internal BamHl site (see below); its
sequence is presented in SEQ ID NO:5. The sequence of primer V28degf2
corresponds to DNA encoding the second intracellular loop region of the

10 canonical receptor structure. See Probst et al, supra. The 3' primer,
designated V28degr2, contains an internal HindUI site (see below); its
sequence is presented in SEQ ID NO:6. The sequence of primer V28degr2
corresponds to DNA encoding the sixth transmembrane domain of the
canonical receptor structure.

15 Amplified PCR DNA was subsequently digested with BamHI

and HindlH to generate fragments of approximately 390 bp, consistent with
the fragment size predicted from inspection of the canonical sequence.
Following endonuclease digestion, these PCR fragments were cloned into
pBluescript (Stratagene Inc., LaJolla, CA). A total of 54 cloned fragments

20 were subjected to automated nucleotide sequence analyses. In addition to
sequences from CCCKR1 and CCCKR2, sequences from the two novel
chemokine receptor genes of the invention were identified. These two novel
chemokine receptor genes were designated 88-2B and 88C.

Restriction endonuclease mapping and hybridization . were

25 utilized to map the relative positions of genes encoding the receptors 88C, 88-
2B. CCCKR1, and CCCKR2. These four genes are closely linked, as the
gene for 88C is approximately 18 KBP from the CCCKR2 gene on human
chromosome 3p21.

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Example 2

Full-length 88-2B and 88C cDNAs were isolated from a
macrophage cDNA library by the following procedure. Initially, a cDNA
library, described in Tjoelker et aL, Nature J74/549-553 (1995), was
5 constructed in pRc/CMV (Irivitrogen Corp., San Diego, CA) from human
macrophage mRNA. The cDNA library was screened for the presence of 88-
2B and 88C cDNA clones by PCR using unique primer pairs corresponding
to 88-2B or 88C. The PCR protocol involved an initial denaturation at 94°C
for four minutes. Polynucleotides were then amplified using 33 cycles of

10 PCR under the following conditions: Denaturation at 94°C for one minute,
annealing at 55' J C for one minute, and extension at 72 U C for two minutes.
The first primer specific for 88-2B was primer 88-2B-fl ; presented in SEQ ID
NO: 11. It corresponds to the sense strand of SEQ ID NO:3 at nucleotides
844-863. The second PCR primer specific for the gene encoding 88-2B was

15 ' primer 88-2B-rl, presented in SEQ ID NO: 12; the 88-2B-rl sequence
corresponds to the anti- sense strand of SEQ ID NO:3 at nucleotides 1023-
1042. Similarly, the sequence of the first primer specific for the gene
encoding 88C. primer 88C-fl , is presented in SEQ ID NO: 13 and corresponds
to the sense strand of SEQ ID NO:l at nucleotides 453-471. The second

20 primer specific for the gene encoding 88C is primer 88C-r3, presented in SEQ
ID NO: 14; the, sequence of 88C-r3 corresponds to the anti-sense strand of
SEQ ID NO: 1 at nucleotides 744-763.

The screening identified clone 777, a cDNA clone of 88-2B. :
Clone 777 contained a DNA insert of 1915 bp including the full length coding

25 sequence of 88-2B as determined by the following criteria: the clone contained
a long open reading frame beginning with an ATG codon, exhibited a Kozak
sequence, and had an in-frame stop codon upstream. The DNA and deduced
amino acid sequences of the insert of clone 777 are presented in SEQ ID
NO: 3 and SEQ ID NO:4, respectively. The 88-2B transcript was relatively

30 rare in the macrophage cDNA library. During the library screen, only three
88-2B clones were identified from an estimated total of three million clones.

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Screening for cDNA clones encoding the 88C chemokine
receptor identified clones 101 and 134 which appeared to contain the entire
88C coding region, including a putative initiation codon. However, these
clones lacked the additional 5' sequence needed to confirm the identity of thb
5 initiation codon. The 88C transcript was relatively abundant in the
macrophage cDNA Library. During the library screen, it was estimated that
88C was present at one per 3000 transcripts (in a total of approximately three
million clones in the library).

RACE PCR (Rapid Amplification of cDNA Ends) was

10 performed to extend existing 88C clone sequences, thereby facilitating the
accurate characterization of the 5' end of the 88C cDNA. Human spleen 5'- '
RACE-ready cDNA was purchased from Clontech Laboratories, Inc. ; Palo
Alto, CA ? and used according to the manufacturer's recommendations. The
cDNA had been made "5' -RACE-ready" by ligating an anchor sequence to the

15 . 5 ; ends of the cDNA fragments.. The anchor sequence is complementary to
an anchor primer supplied by Clontech Laboratories, Inc., Palo Alto, CA.
The anchor sequence-anchor primer duplex polynucleotide contains an EcoSl
site. Human spleen cDNA was chosen as template DNA because Northern
blots had revealed that 88C was expressed in this tissue. The PCR reactions

20 were initiated by denaturing samples at 94°C for four minutes. Subsequently,
-sequences were amplified using 35 cycles involving denaturation at 94°C for
one minute, annealing at 60°C for 45 seconds, and extension at 72°C for two
minutes. The first round of PCR was performed on reaction mixtures
containing 2^1 of the 5 '-RACE-ready spleen cDNA, 1 /xl of the anchor

25 primer, and 1 of primer 88c-r4 (100 ng/id) in a total reaction volume of 50
id. The 88C-specific primer, primer 88c-r4 (5'-GATAAGCCTCACAG-
CCCTGTG-3'), is presented in SEQ ID NO:7. The sequence of primer 88c-
r4 corresponds to the anti-sense strand of SEQ ID NO:l at nucleotides 745-
765. A second round of PCR was performed on reaction mixtures including

30 1 pi of the first PCR reaction with 1 /xl of anchor primer and 1 /xl of primer
88C-rlb (100 ng//xl) containing the following sequence

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(5 ' -GCTAAGCTTG ATG ACTATCTTTAATGTC-3 ' ) and presented in SEQ
ID NO:8. The sequence of primer 88C-rIb contains an internal HindUL
cloning site (underlined). The sequence 3' of the HindUl site corresponds to
the anti-sense strand of SEQ ID NO:l at nucleotides 636-654. The resulting
5 PCR product was digested with EcdBl and HindUL and fractionated on a 1 %
agarose gel. The approximately 700 bp fragment was isolated and cloned into
pBluescript. Clones with the largest inserts were sequenced. Alternatively,
the intact PCR product was ligated into vector pCR using a commercial TA
cloning kit (Invitrogen Corp., San Diego, CA) for subsequent nucleotide

10 sequence determinations.

The 88-2B and 88C cDNAs were sequenced using the PRISM™
Ready Reaction DyeDeoxy"* Terminator Cycle Sequencing Kit (Perkin Elmer
Corp., Foster City, CA) and an Applied Biosystems 373A DNA Sequencer.
The insert of clone 777 provided the double-stranded template for sequencing

15 reactions used to determine the 88-2B cDNA sequence. The sequence of the
entire insert of clone 777 was determined and is presented as the 88-2B cDNA
sequence and deduced amino acid sequence in SEQ ID NO: 3. The sequence
is 1915 bp in length, including 361 bp of 5- untranslated DNA (corresponding
to SEQ ID NO: 3 at nucleotides 1-361), a coding region of 1065 bp

20 (corresponding to SEQ ID NO:3 at nucleotides 362-1426), and 489 bp of 3'
untranslated DNA (corresponding to SEQ ID NO: 3 at nucleotides 1427-1915).
The 88-2B genomic DNA, described in Example 1 above, corresponds to SEQ
ID NO:3 at nucleotides 746-1128. The 88C cDNA sequence, and deduced
amino acid sequence, is presented in SEQ ID NO:l. The 88C cDNA

25 sequence is a composite of sequences obtained from RACE-PCR cDNA, clone
134, and clone 101. The RACE-PCR cDNA was used as a sequencing
template to determine nucleotides 1-654 in SEQ ID NO:l, including the
unique identification of 9 bp of 5' untranslated cDNA sequence in SEQ ID
NO: 1 at nucleotides 1 -9. The sequence obtained from the RACE PCR cDNA

30 confirmed the position of the first methionine codon at nucleotides 55-57 in
SEQ ID NO:l, and supported the conclusion that clone 134 and clone 101
contained full-length copies of the 88C coding region. Clone 134 contained

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45 bp of 5' untranslated cDNA (corresponding to SEQ ID NO: 1 at nucleotides
10-54), the 1056 bp 88C coding region (corresponding to SEQ ID NO:l at
nucleotides 55-1110), and 492 bp of 3' untranslated cDNA (corresponding to
SEQ ID NO:l at nucleotides 1111-1602). Clone 101 contained 25 bp of 5' .
5 untranslated cDNA (corresponding to SEQ ID NO: 1 at nucleotides 30-54), the
1056 bp 88C coding region (corresponding to SEQ ID NO: 1 at nucleotides 55-
1 1 1 0) , and 2273 bp of 3 ' untranslated cDNA (corresponding to SEQ ID NO: 1 .
at nucleotides 1111-3383). The 88C genomic DNA described in Example 1
above, corresponds to SEQ ID NO: 1 at nucleotides 424-809.

10 The deduced amino acid sequences of 88-2B and 88C revealed

hydrophobicity profiles characteristic of GPCRs, including seven hydrophobic
domains corresponding to GPCR transmembrane domains. Sequence
comparisons with other GPCRs also revealed a degree of identity.
Significantly, the deduced amino acid sequences of both 88-2B and 88C had

15 highest identity with the sequences of the chemokine receptors. Table 1
presents the results of these amino acid sequence comparisons. ,

Table 1

Chemokine Receptors

88-2B

88C

IL-8RA

30%

IL-8RB

31%

30%

CCCKR1

62%

54%

CCCKR2A

46%

66%

CCCKR2B

50%

72% .

88-2B

100%

50%

88-C

50%

100%

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Table 1 shows that 88-2B is most similar to CCCKR1 (62 % identical at the
amino acid level) and 88C is most similar to CCCKR2 (72% identical at the
amino acid level).

The deduced amino acid sequences of 88-2B and 88C also
5 reveal the intracellular and extracellular domains characteristic of GPCRs.
The 88-2B extracellular domains correspond to the amino acid sequence
provided in SEQ ID NO:3, and SEQ ID NO:4, at amino acid residues 1-36,
93-107, 171-196, and 263-284. The extracellular domains of 88-2B are
encoded by polynucleotide sequences corresponding to SEQ ID NO: 3 at

10 nucleotides 362-469, 638-682, 872-949, and 1148-1213. Extracellular .
domains of 88C include amino acid residues 1-32, 89-112, 166-191, and. 259-
280 in SEQ ID NO: 1 and SEQ ID NO:2. The 88C extracellular domains are
encoded by polynucleotide sequences that correspond to SEQ ID NO:l at
nucleotides 55-150, 319-390, 550-627, and 829-894. The intracellular

15 domains of 88-2B include amino acids 60-71, 131-151, 219-240, and 306-355
of SEQ ID NO:3 and SEQ ID NO:4. Those domains are encoded by
polynucleotide sequences corresponding to SEQ* ID NO:3 at nucleotides 539-
574, 752-814, 1016-1081 , and 1277-1426, respectively. The 88C intracellular
domains include amino acid residues 56-67, 125-145, 213-235, and 301-352

20 of SEQ ID NO: 1 and SEQ ID NO:2. The intracellular domains of 88C are
encoded by polynucleotide sequences corresponding to SEQ ID NO:l at
nucleotides 220-255, 427-489, 691-759, and 955-1110.

In addition, a macaque 88C DNA was amplified by PGR from
macaque genomic DNA using primers corresponding to 5' and 3' flanking

25 regions of the human 88C cDNA. The 5' primer corresponded to the region
immediately upstream of and including the initiating Met codon. The 3'
primer was complementary to the region immediately downstream of the
termination codon. The primers included restriction sites for cloning into
expression vectors. The sequence of the 5' primer was

30 gac aagctt cacagggtggaacaagaTG (With the HindlH site underlined)
(SEQ ID NO: 17) and the sequence of the 3' primer was
GTC TCTAGA CCACTTGAGTCCGTGTCA (with the Xbal site underlined) (SEQ ID

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NO: 18). The conditions of the PCR amplification were 94 °C for eight
■ minutes, then 40 cycles of 94 °C for one minute, 55 °C for forty-five seconds,
and 72°C one minute. The amplified products were cloned into the HindHI
and Xbal sites of pcDNA3 and a clone was obtained and sequenced. The full
5 length macaque cDNA and deduced amino acid sequences are presented in
SEQ ID NOs: 19 and 20, respectively. The nucleotide sequence of macaque
88C is 98% identical to the human 88C sequence. The deduced amino acid
sequences are 97% identical.

Example 3

10 The mRNA expression patterns of 88-2B and 88C were

determined by Northern blot analyses.

Northern blots containing immobilized poly A* RNA from a
variety of human tissues were purchased from Clontech Laboratories, Inc..
Palo Alto, CA. In particular, the following tissues were examined: heart,
15 brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, .
prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes.

A probe specific for 88-2B nucleotide sequences was generated
from cDNA clone 478. The cDNA insert, in clone 478 contains sequence
corresponding to SEQ ID NO: 3 at nucleotides 641-1915. To generate a
20 probe, clone 478 was digested and the insert DNA fragment was isolated
following gel electrophoresis. The isolated insert fragment was then
radiolabeled with 32 P~labeled nucleotides, using techniques known in the art.

A probe specific for 88C nucleotide sequences was generated
by isolating and- radiolabeling the insert DNA fragment found in clone 493.
25 The insert fragment from clone 493 contains sequence corresponding to SEQ
ID NO: 1 at nucleotides 421-1359. Again, conventional techniques involving
32 P-labeled nucleotides were used to generate the probe.

Northern blots probed with 88-2B revealed an approximately
1 . 8 kb mRNA in peripheral blood leukocytes. The 88C Northerns showed an
30 approximately 4 kb mRNA in several human tissues, including a strong signal
when probing spleen or thymus tissue and less intense signals when analyzing

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' - 22 - ; ' -

mRNA from peripheral blood leukocytes and small intestine. A relatively
weak signal for 88C was detected in lung tissue and in ovarian tissue.

The expression of 88C in human T-cells and in hematopoietic
cell lines was also determined by Northern blot analysis. Levels of 88C in
5 C!D4 + and CD8 + T-cells were very high. The transcript was present at
relatively high levels in myeloid cell lines THP1 and HL-60 and also found
in the B cell line Jijoye. In addition, the cDNA was a relatively abundant .
.transcript in a human macrophage cDNA library based on PCR amplification
of library subtractions.

10 Example 4

. The 88-2B and 88C cDNAs were expressed by recombinant
methods in mammalian cells.

For transient transfection experiments, 88C was subcloned into
the mammalian cell expression vector pBJl (Ishi, K. et, aL, J. Biol. Chem

15 270:16435-16440 (1995). The construct included sequences encoding a
prolactin signal sequence for efficient cell surface expression and a FLAG
epitope at the amino terminus of 88C to facilitate detection of the expressed
protein. The FLAG epitope consists of the sequence "DYKDDDD. " COS-7
cells were transiently transfected with the 88C expression plasmid using

20 •. * Lipofectamine (Life Technology, Inc. , Grand Island, NY) following the
. manufacturer's instructions. Briefly, cells were seeded in 24-well plates at a
density of 4 X 10 4 cells per well and grown overnight. The cells were then
washed with PBS, and 0.3 mg of DNA mixed with 1.5 ^1 of lipofectamine in
0.25 ml of Opti-MEM was added to each well. After 5 hours at 37°C. the

25 . medium was replaced with medium containing 10% FCS. quantitative ELISA
confirmed that 88C was expressed at the cell surface in transiently transfected

COS-7 cells using the Ml antibody specific for the FLAG epitope (Eastman

*s ■

Co., New Haven, CT).

The FLAG-tagged 88C receptor was also stably transfected into
30 HEK-293 cells, a human embryonic kidney cell line, using transfection
reagent DOTAP (N-[l-[(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyI-

PCT/US96/20759

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ammoniumrnethylsulfate.Boehringer-Mannheim, Inc. Indianapolis, IN)
according to the manufacturer' s recommendations. Stable lines were selected
in the presence of the drug G418. The transfected HEK-293 cells were
evaluated for expression of 88C at the cell surface by ELISA. using the Ml
5 antibodv to the FLAG epitope. ELISA showed that 88C tagged with the
FLAG epitope was expressed at the cell surface of stably transformed HEK-
293 Cells.

The 88-2B and 88C cDNAs were used to make stable HEK-293
transfectants. The 88-2B receptor cDNA was cloned behind the cytomegalovi-
10 rus promoter in pRc/CMV (Invitrogen Corp., San Diego, CA) using a PCR-
based strategy. The template for the PCR reaction was the cDNA insert in
clone 777. The PCR primers were 88-2B-3 (containing an internal Xbal site)
: and 88-2B-5 (containing an internal //mdffl site). The nucleotide sequence of
primer 88-2B-3 is presented in SEQ ID NO:9; the nucleotide sequence of
15 primer 88-2B-5 is presented in SEQ ID NO: 10. An 1 104 bp region of cDNA ■
was amplified. Following amplification, the DNA was digested with Xbal and
HinWl and cloned into similarly digested P Rc/CMV. The resulting plasmid
was named 777XP2, which contains 18 bp of 5' untranslated sequence, the
entire coding region of 88-2B, and 3 bp of 3'. untranslated sequence. For the
20 88C sequence, the fulMength cDNA insert in clone 134 was not further
modified before transfecting HEK-293 cells.

To create stably transformed cell lines, the pRc/CMV
■ recombinant clones were transfected using transfection reagent DOTAP (N-[l -
[( 2.3-Dioleoyloxy)propyl]-N,N,N-trimethyl-

25 ammoniummethylsulfate.Boehringer-Mannheim, Inc., IndianapoUs, IN)
according to the manufacturer's recommendations, into HEK-293 cells, a
human embryonic kidney cell line. Stable lines were selected in the presence
of the drug G418. Standard screening procedures {i.e., Northern blot
analyses) were performed to identify stable cell lines expressing the highest

30 levels of 88-2B and 88C mRNA.

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Example 5

A. Ca^ Flux Assays

To analyze polypeptide expression, a functional assay for
chemokine receptor activity was employed. A common feature of signalling
5 through the known chemokine receptors is that signal transduction is
associated with the release of intracellular calcium cations. Therefore,
intracellular Ca ++ concentration in the transfected HEK-293 cells was assayed
to determine whether the 88-2B or 88C receptors responded to any of the
known chemokines.

10 HEK-293 cells, stably transfected with 88-2B, 88C (without the

FLAG epitope sequence), or a control coding region (encoding IL8R or
CCCKR2, see below) as described above, were grown in T75 flasks to
approximately 90% confluence in MEM + 10% serum. Cells were then
washed, harvested with versene (0.6 mM EDTA, 10 mM Na 2 HP0 4 , 0.14 M
15 NaCl, 3 mM KC1, and 1 mM glucose), and incubated in MEM +10%. serum
-h 1 M M Fura-2 AM (Molecular Probes, Inc., Eugene, OR) for 30 minutes
at room temperature. Fura-2 AM is a Ca ++ -sensitive dye. The cells were
resuspended in Dulbecco's phosphate-buffered saline containing 0.9 mM CaCl 2
and 0.5 mM MgCl 3 (D-PBS) to a concentration of approximately 10 7 cells/ml
20 and changes in fluorescence were monitored using a fluorescence.

spectrophotometer (Hitachi Model F-4010). Approximately 10 6 cells were "
suspended in 1.8 ml D-PBS in a cuvette maintained at 3TC. Excitation
wavelengths alternated between 340 and 380 nm at 4 second intervals; the
emission wavelength was 510 nm. Test compositions were added to the
25 cuvette via an injection port; maximal Ca ++ flux was measured upon the
addition of ionomycin.

Positive responses were observed in cells expressing IL-8RA
when stimulated with IL-8 and also when CCCKR2 was stimulated with MCP-
1 or MCP-3. However, HEK-293 cells expressing either 88-2B or 88C failed
30 to show a flux in intracellular Ca ++ concentration when exposed to any of the
following chemokines: MCP-1, MCP-2, MCP-3, MTP-la, MIP-1/3, IL8,

PCT/US96/20759

WO 97/22698

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NAP-2, gro/MGSA, DP-10, ENA-78, or PF-4. (Peprotech, Inc., Rocky Hill,

NJ)

Using a more sensitive assay, a Ca" flu* response to RAOTES
was observed microsoopicaily in Fura-2 AM-loadfcd cells expressing 88-2B..

5 The assay involved cells and reagents prepared as described above. RANTES
(Regulated on Activation, tjormal 1 Expressed and Secreted) is a CC
chemokine that has been identified as a chemoattractant and activator of
eosinophils. See Neo,e e, al, supra. This chemokine also mediates the
re ,ease of histamine by basophils and has been shown to functton as a

,0 chemoat^ctan, for memory T cells in *ro. Modulation of 88-2B receptor
activities is therefore contemplated to be useful in modulating leukocyte
activation.

FLAG tagged 88C receptor was expressed in HEK-293 cells
and tested for chemokine interactions in the CA- flux assay. Cell surface
15 expression of 88C was confirmed by EUSA and by FACScan analysis ustng
the Ml antibody. The chemokines RANTES, MW-1«, and MlP-lrS all
induced a Ca~ flux in 88C-transfected cells when added at a concentrate
of 100 nM.

Ca^ + flux assays can also be designed to identify modulators of
,0 chemokine receptor binding. The preceding fluorimetric or microscopic
assays are carried out in the presence of test compounds. If Ca- flux is
increased in the presence of a test compound, that compound is an actxvator
of chemokine receptor binding. In contrast, a diminished Cm- flux identifies
the test compound as an inhibitor of chemokine receptor bindmg.

25 g Phos phoin ^itnl Hydrolysis

Another assay for ligands or modulators involves monitoring
phospholipase C activity, as described in Hung ei al, J. Biol. Chem. 116:911-
832 (1992). initially, host cells expressing a chemokine receptor are loaded
with 'H-inositol for 24 hours. Test compounds (i.e., potential ligands) are
30 then added to the cells and incubated at 3TC for 15 minutes. The cells are
then exposed to 20 mM formic acid to solubilize and extract hydrolyzed

WO 97/22698 PCT/US96/20759

-26-

metabolites of phosphoinositol metabolism (/. e. , the products of phospholipase
C-mediated hydrolysis). The extract is subjected to anion exchange
chromatography using an AG 1X8 anion exchange column (formate form).
Inositol phosphates are eluted with 2 M ammonium formate/0. 1 M formic acid
5 and the 3 H associated with the compounds is determined using liquid
scintillation spectrophotometry. The phospholipase C assay can also be
exploited to identify modulators of chemokine receptor activity. The
aforementioned assay is performed as described, but with the addition of a
potential modulator. Elevated levels of detectable label would indicate the
10 modulator is an activator; depressed levels of the label would indicate the
modulator is an inhibitor of chemokine receptor activity.

The phospholipase C assay was performed to identify
chemokine ligands of the FLAG-tagged 88C receptor. Approximately 24
hours after transfection, COS-7 cells expressing 88C were labeled for 20-24
15 hours with myc?-[2- 3 H]inositol (1 /xCi/ml) in inositol-free medium containing
10% dialyzed FCS. Labeled cells were washed with inositol-free DMEM
containing 10 mM LiCl and incubated at 37 °C for 1 hour with inositol-free
DMEM containing 10 mM LiCl and one of the following chemokines:
RANTES, MIP-1/?, MIP-Iq:, MCP-1, IL-8, or the murine MCP-1 homolog
20 JE. Inositol phosphate (IP) formation was assayed as described in the
previous paragraph. After incubation with chemokines, the medium was
aspirated and cells were lysed by addition of 0.75 ml of ice-cold 20 mM
formic acid (30 min). Supernatant fractions were loaded onto AG1-X8 Dowex
columns (Biorad. Hercules, CA), followed by immediate addition of 3 ml of
25 50 mM NH 4 OH. The columns were then washed with 4 ml of 40 mM
ammonium formate, followed by elution with 2 M ammonium formate. Total
inositol phosphates were quantitated by counting beta-emissions.

Because it has been shown that some chemokine receptors, such
as IL8RA AND IL8RB, require contransfection with an exogenous G protein
30 before signalling can be detected in COS-7 cells, the 88C receptor was co-
expressed with the chimeric G protein Gqi5 (Conklin, et aL t Nature 363:274-
276. (1993). Gqi5 ia a G protein which has the carboxyl terminal five amino

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- 27 -

acids of Gi (which bind to the receptor) spliced onto Gaq. Co.-transfection
with Gqi5 significantly potentiates signaling by CCCKR1 and CCKR2B. Co-
transfection with Gqi5 revealed that 88C signaled well in response to
RANTES, MIP- 1/3, and MHMor, but not in response to MCP-1, IL-8 or the
5 murine MCP-1 homologue JE. Dose-response curves revealed EC 50 values of
InM for RANTES, 6nM for MIP-10, and 22nM for MTP-la.

88C is the first cloned human receptor with a signaling response
to MIP- 10. Compared with other CC chemokines, MIP- 1/3 clearly has a
unique cellular activation pattern. It appears to activate T cells but not

10 monocytes (Baggiolini et al., Supra) which is consistent with receptor
stimulation studies. For example, while MIP- 1/3 binds to CCCKRl ? -it does
not induce calcium' flux (Neote et al., Supra). In contrast, MIP- la and
RANTES bind to and causes signalling in CCCKR1 and CCCKR5 (RANTES
also causes activation of CCCKR3). MIP- 1/3 thus appears to be much more

15 . selective than other chemokines of the CC chemokine family. Such selectivity
is of therapeutic significance because a specific beneficial activity can be
stimulated (such as suppression of HIV infection) without stimulating multiple
leukocyte populations which results in general pro-inflammatory activities.

C. - BINDING ASSAYS

20 Another assay for receptor interaction -with chemokines was a

modification of the binding assay described by Ernst et al. J. Immunol.
752:3541-3549 (1994). MIP- 1/3 as labeled using the Bolton and Hunter
reagent (di-iodide, NEN, Wilmington, DE), according to the manufacturer's
instructions. Unconjugated iodide was separated from labeled protein by

25 elution using a PD-10 column (Pharmacia) equilibrated with PBS and BSA
(1 % w/v). The specific activity was typically 2200 Ci/mmole. Equilibrium
binding was performed by adding 125 I-labeled ligand with or without a 100-
fold excess of unlabeled ligand, to 5 X 10 5 HEK-293 cells transfected with
88C tagged with the FLAG epitope in polypropylene tubes in a total volume

30 of 300 ii\ (50 mM HEPES pH 7.4, 1 mM CaCl 2 , MgCl 2; 0.5% BSA) and
incubating for 90 minutes at 27 °C with shaking at 150 rpm. The cells were

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- 28 -

collected, using a Skatron cell harvester (Skatron Instruments Inc., Sterling,
VA), on glass fiber filters presoaked in 0.3% polyethyleneimine and 0.2%
BSA. After washing, the filters were removed and bound ligand was
quantitated by counting gamma emissions. Ligand binding by competition
5 with unlabeled ligand was determined by incubation of 5 X 10 5 transfected
cells (as above) with 1.5 nM of radiolabeled ligand and the indicated
concentrations of unlabeled ligand. The samples were collected, washed and
counted as above. The data was analyzed using the curve-fitting program
Prism (GraphPad Inc., San Diego, CA) and the iterative non-linear regression

10 program, LIGAND (PM220).

In equilibrium binding assays, 88C receptor bound radiolabeled
MlP-IjS in a specific and saturable manner. Analysis of this binding data by
the method of Scatchard revealed a dissociation constant (Kd) of 1.6 nM.
Competition binding assays using labeled MIP-1/3 revealed high-affinity

15 binding of MIP-1/? (IC 50 = 7.4 nM), RANTES (IC 50 = 6.9 nM), and MIP-1 a
(IC 50 = 7.4 nM), consistent with the signaling data obtained in transiently
transfected COS-7 cells as discussed in section B above.

Example 6

The chemokines MIP-la, MIP-lj3 and RANTES have been
20 shown, to inhibit replication of HIV- 1 and HIV-2 in human peripheral blood
mononuclear cells and PM1 cells (Cocchi, et. aL, supra). In view of this
finding and in view of the results described in Example 5, the present
invention contemplates that activation of or ligand binding to the 88C receptor
may provide a protective role in HIV infection.
25 Recently, it has been reported that the orphan G protein-coupled

receptor, fusin, can act as a co-receptor for HIV entry. Fusin/CXCR4 in
combination with CD4, the primary HIV receptor, apparently facilitates HIV
infection of cultured T cells {Feng, et al , Science 272:872-877 (1996). Based
upon the homology of fusin to chemokine receptors and the chemokine
30 binding profile of 88C, and because 88C is constitutively expressed in T cells

WO 97/22698 PCT/US96/20759

-29-

and abundantly expressed in macrophages, 88C is likely to be involved in
viral and HIV infection.

The function of 88C and 88-2B as co-receptors for HIV was
determined by transfecting cells which express CD4 with 88C or 88-2B and
5 challenging the co-transfected cells with HIV. Only cells expressing both
CD4 and a functional co-receptor for HIV become infected. HIV infection
can be determined by several methods. ELISAs which test for expression of
HIV antigens are commercially available, for example Coulter HIV-1 P 24
antigen assay (US Patent Nos. 4,886,742), Coulter Corp., 11800 SW 147th

10 Ave., Miami, FL 33196. Alternatively, the test cells can be engineered to
express a reporter gene such as LACZ attached to the HIV LTR promoter
[Kimpton et al. . J. Virol. 66:2232-2239 (1992)]. In this method, cells that are
infected with HIV are detected by a colorimetric assay.

88C was transiently transfected into a cat cell line, CCC

15 [Clapham, et aL, 757:703-715 (1991)], which had been stably tranformed to
express human CD4 (CCC-CD4). These cells are normally resistant to
infection by any strain of HIV-1 because they do not endogenously express
88C. In these experiments, CCC/CD4 cells were transiently transfected with
88C cloned into the expression vector pcDNA3.1 (Invitrogen Corp., San

20 Diego, CA) using lipofectamine (Gibco BRL, Gaithersburg, MD). Two days
after transfection, cells were challenged with HIV. After 4 days of
incubation, cells were fixed and stained for p24 antigen as a measure of HIV
infection. 88C expression by these cells rendered them susceptible to
infection by several strains of HIV-1. These strains included four primary

25 non-syncytium-inducing HIV-1 isolates (M23, E80, SL-2 and SF-162) which
were shown to use only 88C as a co-receptor but not fusin. Several primary
syncytium-inducing strains of HIV-1 (2006, M13, 2028 and 2076) used either
88C or fusin as a co-receptor. Also, two established clonal HIV-1 viruses
(GUN-1 and 89.6) used either 88C or fusin as a co-receptor.

30 It has been reported that some strains of HIV-2 can infect

certain CD4-negative cell lines, thus implying a direct interaction of HTV-2
with a receptor other than CD4 [Clapham, et aL, J. ViroL 66:3531-3537

WO 97/22698 PCT/US96/20759

- 30 -

(1992)] For some strains of HIV-2, this infection is facilitated by the presence
of soluble CD4 (sCD4). Since 88-2B shares high sequence similarity with
other chemokine receptors that act as HIV co-receptors (namely 88C and
fusin). 88-2B was considered to be a likely HIV-2 co-receptor. The role of
5 88-2B as an HIV-2 co-receptor was demonstrated using HIV-2 strain ROD/B.
Cat CCC cells which do not eridogeneously express CD4 were transfected
with 88-2B. In these experiments, cells were transfected with pcDNA3.1
containing 88-2B using lipofectamine and infected with HIV-2 48 hours later.
Three days after infection, cells were immunostained for the presence of HIV -

10 2 envelope glycoproteins. The presence of sCD4 during HIV-2 ROD/B challenge
. increased the infection of these cells by by 10-fold. The entry of HIV-2 into
the 88-2B transfected cells could be blocked by the presence of 400-800 ng/ml
eotaxin, one of the ligands for 88-2B. The baseline infectivity levels of
CCC/88-2B (with no soluble CD4) were equivalent to CCC cells which were

15 not transfected with 8 8-2B.

The role of 88-2B and 88C as co-receptors for HTV was
confirmed by preparing and challenging cell lines stably transformed to
express 88C or 88-2B with various strains of HIV and SIV. These results are
described in Example 7.

20 Alternatively, the co-receptor role of 88C and 88-2B can be

demonstrated by an experimental method which does not require the use of
live virus. In this method, cell lines co-expressing 88C or 88-2B, CD4 and
a LACL reporter gene are mixed with a cell line co-expressing the HTV
envelope glycoprotein (KNV) and a transcription factor for the reporter gene

25 construct (Nussbaum, et aL, 1994 7. Virol. 65:5411). Cells expressing a
functional co- receptor for HTV will fuse with the ENV expressing cells and
thereby allow expression of the reporter gene. In this method, detection of
reporter gene product by colorimetric assay indicates that 88C or 88-2B
function as a co-receptor for HTV.

30 The mechanism by which chemokines inhibit viral infection has

not yet been elucidated. One possible mechanism involves activation of the
receptor by binding of a chemokine. The binding of the chemokine leads to

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- 31 -

signal transduction events in the ceil that renders the cell resistant to viral
infection and/or prevents replication of the virus in the cell. Similar to
interferon induction, the cell may differentiate such that it is resistant to viral
infection, or an antiviral state is established. Alternatively, a second
5 mechanism involves direct interference with viral entry into cells by blocking
access of viral envelope glycoproteins to the co-receptor by chemokine
binding. In this mechanism, G-protein signalling is not required for
chemokine suppression of HIV infection.

To distinguish between two mechanisms by which 88C or 88-2B
10 may function as co-receptors for viral or HIV infection, chemokine binding
to the receptor is uncoupled from signal transduction and the effect of the
chemokine on suppression of viral infection is determined.

Ligand binding can be uncoupled from signal transduction by
the addition of compounds which inhibit G-protein mediated signaling. These
15 compounds include, for example, pertussis toxin and cholera toxin. In
addition, downstream effector polypeptides can be inhibited by other
compounds such as wortmannin. If G-protein signalling is involved in
. suppression of viral infection, the addition of such compounds would prevent
suppression of viral infection by the chemokine. Alternatively, key residues
20 or receptor domains of 88C or 88-2B receptor required for G-protein coupling
can be altered or deleted such that G-protein coupling is altered or destroyed
but chemokine binding is not affected.

Under these conditions, if chemokines are unable to suppress
viral or HIV infection, then signaling through a G-protein is required for
25 suppression of viral or HIV infection. If however, chemokines are able to
suppress viral infection, then G-protein signaling is not required for
chemokine suppression of viral infection and the protective effects of
chemokines may be due to the chemokine blocking the availability of the
receptor for the virus.
30 Another approach involves the use of antibodies directed against

* 88C or 88-2B. Antibodies which bind to 88C or 88-2B which can be shown
not to elicit G-protein signaling may block access to the chemokine or viral

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' . - 32 -

binding site of the receptor. If in the presence of antibodies to 88C or 88-2B.
viral infection is suppressed, then the mechanism of the protective effects of
chemokines is blocking viral access to its receptor. Feng, et al. Reported that
antibodies to the amino terminus of the fusin receptor suppressed HIV
5 infection (Feng, et aL, 1996).

Example 7

Cell lines were stably transformed with 88C or 88-2B to further
delineate the role of 88C and 88-2B in HIV infection. Kimpton and
Emerman, "Detection of Replication-Competent and Pseudotyped Human

10 Immunodeficiency Virus with a Sensitive Cell Line on the Basis of Activation
of an Integrated Beta-Galactosidase Gene," J, Virol, 66(5): 3026-3031 (1992)
previously described an indicator cell line, herein identified as HeLa-MAGI
cells. HeLa-MAGI cells are HeLa cells that have been stably transformed to
express CD4 as well as integrated HIV-1 LTR which drives expression of a

15 nuclear localized /S-galactosidase gene. Integration of an HIV provirus in the
cells leads to production of the viral transactivator, Tat, which then turns on
expression of the /3-galactosidase gene. The number of cells that stain positive
with X-gal for £-galactosidase activity in situ is directly proportional to the
number of infected. cells.

20 These HeLa-M AGI cells can detect lab-adapted isolates of HIV-

1 but only a minority of primary isolates [Kimpton and Emerman, supra] , and
cannot detect most SIV isolates [Chackerian et al. 9 "Characterization of a
CD4-Expressing Macaque Cell Line that can Detect Virus After A Single
Replication Cycle and can be infected by Diverse Simian Immunodeficiency

25 Virus Isolates." Virology, 21 3 (2): 6499 -6505 (1995)]..

In addition. Harrington and Geballe, "Co-Factor Requirement
. for Human Immunodeficiency Virus Type 1 Entry into a CD4-Expressing
Human Cell Line, 7. Virol. , (57:5939-5947 (1993) described a ceil line based
on U373 cells -that had been engineered to express CD4 and the same LTR-/3-

30 galactosidase construct. It was previously shown that this cell line, herein
identified as U373-MAGI* could not be infected with any HTV (M or T-tropic)

WO 97/22698 , PCT/US96/20759

- 33 - .

strain of HIV, but could be rendered susceptable to infection by fusion with
HeLa cells [Harrington and Geballe, supra].

In order to construct indicator cell lines that could detect either
Macrophage or T cell tropic viruses, epitope-tagged 88C or 88-2B encoding
5 DNA was transfected into HeLa-MAGI or U373-MAGI cells by infection with
a retroviral vector to generate HeL A-M AGI- 8 8 C or U373-MAGI-88C cell
lines, respectively. Expression of the co-receptors on the cell surface was
demonstrated by immunostaining live cells using the anti-FLAG Ml antibody
and by RT-PCR.

0 The 88C and 88-2B genes utilized to construct HeLa-MAGI -
88C and U373-MAGI-88C included sequences encoding the prolactin sienal
peptide followed by a FLAG epitope as described in Example 4. This gene
was inserted into the retroviral vector pBabe-Puro [Morgenstern and Land
Nucelic Acids Research, 18(1 2):35S7-3596 (1990)]. High titer retroviral .

) vector stocks pseudotyped with the VSV-G protein were made by transient
transfection as described in Bartx et aL % J. ViroL 70:2324-2331 (1996), and
used to infect HeLa-MAGI and U3 73 -MAGI cells. Cells resistant to 0.6
/xg/ml puromycin (HeLa) or 1' /xg/ml puromycin (U373) were pooled. Each
pool contained at least 1000 independent transduction events. An early

1 passage (passage 2) stock of the original HeLa-MAGI cells [Kimpton and
Emerman, supra] was used to create HeLa-MAGI-88C- cells.

Infections of the indicator cell lines with HIV were performed
in 12-well plates with -10-fold serial dilutions of 300 /xl of virus in the
presence of 30 fig/ ml DEAE-Pextran as described [Kimpton and Emerman,
25 supra].

All HTV-1 strains and SIV mac239 were all obtained from the NIH
AIDS Reference and Reagent Program. Molecular clones of primary HIV-
2?3i2A [Gao et al , "Genetic Diversity of Human Immunodeficiency Virus Type
2: Evidence for Distinct Sequence Subtypes with Differences in Vims
30 Biology/' 7. ViroL, 68(1 1)\1 ^33-1 AA1 (1992)] and SIVsmPbjl.9 [Dewhurst
et al, "Sequence Analysis and Acute Pathogenicity of Molecularly Cloned
SIV smm -PBjl4," Nature, 345: 636-640 (1990)] were obtained from B. Hahn

WO 97/22698

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- 34 -

(UAB), All other SIV inne isolates were obtained from Julie Overbaugh (U.
Washington, Seattle). Stocks from cloned proviruses were made by transient
transfection of 293 cells. Other viral stocks were made by passage of virus
in human peripheral blood mononuclear cells or in CEMxl74 cells (for SIV
stocks.) Viral stocks .were normalized by ELISA or p24 gag (Coulter
Immunology) or p27**« (Coulter Immunology) for HTV-1 and fflV-2/SIV,
respectively, using standards provided by the manufacturer.

U373-MAGI-88C cells and U373-MAGI cells (controls) and
were infected with limiting dilutions of a T-tropic strain of HIV-1 (HF/^,
an M-tropic strain (fflV YU . 2 ), and an SIV isolate, SIV ^239.' Infectivity was
measured by counting the number of blue cells per well per volume of virus
(Table 2).

Table 2

virus strain 3

titer on cell line <TU/ml) b

U373-MAGI

U373-MAGI-88C

HIV-1 ^

■ <100

< 100

fflV-l vu . 2

<100

2.2 x 10 6

SIV N1AC 239

1.2 x 10 3

4 x 10 5

a Viruses derived by transfection of molecular clones into 293
cells.

b Infectious units (IU) per ml is the number of blue cells per
well multiplied by the dilution of virus supernatant and
normalized to .1 ml final volume.

Two days after infection, cells were fixed and stained for /?-
galactosidase activity with X-gal. The U373-derived MAGI cells were stained
for 120 minutes at 37°C and the HeLa-derived MAGI cells were stained for
50 minutes at 37°C. Background staining of non-infected cells never
exceeded more than approximately three blue cells per well. Only dark blue
cells were counted, and syncytium with multiple nuclei were counted as a
single infected cell. The infectious titer is the number of blue cells per well
multiplied by the. dilution of virus and normalized to 1 ml. The titer of

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- 35 -

fflV vu . 2 on U373-MAGI-88C cells was 2 x 10 6 . In contrast, the titer of fflV-
1 LA! was less than 100 on U373-MAGI-88C. Thus, the specificity of a
particular HIV strain for 88C varied by four orders of magnitude.

Although SIVMAC239 infection was increased to 4 x 10 5 in
5 U373-MAGI-88C it also clearly infected U373-MAGI cells (Table 2);

Next, a series of primary uncloned HIV strains and cloned M-
tropic strains of HIV-1 were analyzed for their ability to infect indicator cell
lines that express 88C.

As described above, HeLa-MAGI and HeLa-MAGI-88C cells

10 were infected with limiting dilutions of various HTV strains. The two cloned
M-tropic viruses, fflV JR _ CSF and HIV YU . 2) both infected HeLa-MAGI-88C ? but
not HeLa-MAGI cells, showing that both strains use 88C as a co-receptor
(Table 3 ; See note c). However, a great disparity in the ability of each of
these two viral strains to infect HeLa-MAGI-88C cells was observed, 6.2 x

15 10 5 IU/ml for HIV YU . 2 and 1.2 x 10 4 for fflV JR . CSF . The infectivity of virus
stock (Table 3) is the number of infectious units- per physical particle
(represented here by the amount of viral core protein). In addition, it was
observed that the infectivity of these two cloned viral strains differed by over
50-fold in viral stocks that were independently prepared.

20 The variability of infectivity of primary viral isolates was

further examined by analyzing a collection of twelve different uncloned virus
stocks from three different clades (Table 3). Three clade A primary isolates,
three clade E isolates, and three additional clade B isolates from
geographically diverse origins were used. With all nine strains, the primary

25 strains of HIV could be detected on HeLa-MAGI-88C cells, but not on HeLa-
MAGI ceils (Table 3). However, the efficacy of infection varied from five
infectious units per ng p24 gafi to over 100 infectious units per ng p24 eag (table
3). These results indicate that absolute infectivity of M-tropic strains varies
considerably and is independent of clade. A hypothesis that may explain this

30 discrepancy may involve the affinity of the V3 loop of each viral strain for
88C after CD4 binding [Trkola etaL, Nature, 384(6605 'J: 184- 187 (1996); Wu
a aL. Nature, 384(6605 * 179-1 83 (1996)].

WO 97/22698

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Table 3

. virus strain*

viral sub-tvpe
(country of ori2in)°

titer ClU/mJ) on
HeLa-MAGI-88C c

P24 E * lf ns/ml

Infectivitv d

J4TV-1

B (USA)

6.2 x 10*

2200

281

□ TV t

R (USA}

12000

2800

4.2

UTV 1

ni v - j THO20

4133

XJT\7 1

HIV-1 TH0 ,,

4967

T41V 1

Ml V-1 TH022

E (Thailand^

200

HIV-1 USW0

B (USA)

2367

127

HIV-i UG031

A (Uganda) ,

1633

HIV-1 RW009

A (Rwanda)

3333

158

HIV- 1

A (Rwanda)

739

143

5.2

HIV-1 US7:7

B (USA)

14,067

289

HIV-1 US05 ,

B (USA)

5833

284

■ HIV-1^,

B (France)

2.8 x 10 3

167

1600

15 a HTSM YU _ 2 and fflV-l JR ; CSF were derived by transfection of

molecular clones. All others were tested as crude supernatants
of uncloned viral stocks derived from infection of heterologous
peripheral blood mononuclear cells.

b Clade designation according to Myers et al, 1995 for the
20 env gene; country of origin refers to the country of residence

of the HIV-positive individual from whom blood was obtained
for viral isolation (World Health Organization Viral Isolate
Program). -

c Infectious units (IU) per ml is the number of blue cells per
25 well multiplied by the dilution of virus supernatant and

normalized to 1 ml final volume. All viruses, except HIV-1 LA1 ,
had less than 10 IU/ml when tested on HeLa-MAGI cells
without 88C. HTV^j, a T-tropic strain, has a titer of 2.8 x 10 5
on HeLa-MAGI cells with or without 88C.

30 . d Infectivity is the infectious units per ng P24 gag (column four

divided by column five).

The ability of the HeLa-MAGI- 8 8 C cells to detect HIV-2 and.
other SIV strains was also determined. HIV-2 Rod has been reported to use
fusin as a receptor even in the absence of CD4 [Endres et al, Cell,

WO 97/22698

- 37 -

PCT/US96/20759

§7^:745-756 (1996)]. fflV-2 Rod is able to infect HeLa-MAGI cells, however
its infectivity is enhanced at least 10-fold in HeLa-MagI-88C (Table 4). HeLa
cells endogenously express fusin. Thus, the molecular clone of HIV-2 Rod is
dual tropic, and is able to use 88C as one of its co-receptors in addition to
5 CXCR4. Similarly, a primary strain of fflV-2 73I2A infected HeLa-MAGI-88C
cells and not the HeLa-MAGI cells, indicating that like primary strain of HIV -
1 , it uses 88C as a receptor. ■

. Table 4

virus strain J

reference .

titer (IU/ml) on
HeLa-MAGI h

titer (IU/ml) on
HeLa-MAGI- .
88C

" Infectivity on HeLa-
MAGI-88C C

^1Y"-KCJ[>?

(Guyader efaL, 1987)

967

5900

HIV-2 73I2A

(Gzo et al. 1994)

<30

6500

SIV MAC 239

(Naidu et al , 1 988)

<30

20900

-90

SIV MNE cl8

(O verbau gh et at.. 1991 )

<30

15700

SIV MN? 170

(Rudensey et al. , 1995)

< 30

10700

SIV SM PbjI.9

(Dewhurst et al. , 1990)

<30

^ 776

ND d

SIV AOM 9063

(Hirsch et aL , 1995)

<30

< 1

3 fflV-2 stocks. SIV SM Pbjl.9, and SIV AGM 9063 were tested
directly after by transfection of molecular clones in 293 cells.
All others were derived from transfection of molecular clones
20 and subsequently amplified in CEMx 1 74 cells.

b Infectious units (IIP) per ml is the number of blue cells per
well multiplied by the dilution of virus supernatant and /
normalized to 1 ml final volume. All viruses in this panel
were also negative on HeLa-MAGI- 8 8-2B .

25 . c Infectivity is the infectious units (on 88C expressing cells) per

ng P27 pn? determined by ELISA.

d ND, not determined.

None of the SIV strains tested infected the HeLa-MAGI cells
(Table 4), and none infected HeLa-MAGI cells that expresses 8 8-2B. This
30 indicates that an alternative co-receptor used -by SIV in U373 cells is not
expressed in HeLa cells, and is not 88-2B. All SIV strains tested infected the

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HeLa-MAGI-88C cells to some extent (Table 3) indicating that all of the
tested SIV strains use at least 88 C as one of their co-receptors.

The classification of M-tropic and T-tropic strains of HIV in the
past has often been correlated with another designation "non-syncytium
5 inducing" (NSI), and "syncytium inducing" (SI), respectively. Assays based
on the cell lines described herein are sensitive to syncytium formation. The
infected cells can form large and small foci of infection containing multiple
nuclei [Kimpton and Emerman, supra].

Experiments using multiple different viral strains and U373-

10 MAGI-88C or HeLa-MAGI-88C indicate that SI/NSI designation is not
meaningful because all viral strains formed syncytia if the correct co-receptbr
was present. These experiments show that syncytium formation is more likely"
a marker for the presence of an appropriate co-receptor on the infected cell,
rather than an indication of tropism. ^ Infection of the HeLa-MAGI-88C cells

15 with SIV strains reported in the literature to be non-syncytium forming strains,
in particular, SIV MAC 239, SIV^cl 8, and SIV^eITO, was remarkable because
the size of the syncytia induced in the monolayer was much larger than those
induced by any other the HIV strains.

Example 8

20 - Mouse monoclonal antibodies which specifically recognize 88C

were prepared. The antibodies were produced by immunizing mice with a
peptide corresponding to the amino terminal twenty amino acids of 88C. The
peptide was conjugated to Keyhole Limpet Cyanin (KLH) according to the
manufacturer's directions (Pierce, Imject maleimide activated KUH),

25 emulsified in complete Freund's adjuvant and injected into five mice. Two
additional injections of conjugated peptide in incomplete Freund's adjuvant
occurred at three week intervals. Ten days after the final injection, serum
from each of the five mice was tested for immunoreactivity with the twenty
amino acid peptide by ELISA. In addition, the immunoreactivity of the sera

30 were tested against intact 88C receptor expressed on the surface of 293 cells
by fluorescence activated cell sorting (FACS). The mouse with the best anti-.

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; . - 39 - ■* •

88C activity was chosen for spleen cell fusion and production of monoclonal
antibodies by standard laboratory methods. Five monoclonal cell lines (227K,
227M, 227N, 227P, 227R) were established which produced antibodies that
. recognized the peptide by ELISA and the 88C protein on 293 cells by FACS.
5 Each antibody was shown to react only with 88C-expressing 293 cells, but not
■ * with 293 cells expressing the closely related MCP receptor (CCCKR-2). Each
antibody was also shown to recognize 8 8C expressed transiently in COS cells.

Rabbit polyclonal antibodies were also generated against 88C.
Two rabbits were injected with conjugated amino-terminal peptide as described

10 above. The rabbits were further immunized by four additional injections of
the conjugated amino-terminal peptide. Serum from each of the rabbits (2337J
and 2470J) was tested by FACS of 293 cells expressing 88C. The sera
specifically recognized 88C on the surface of 293 cells.

The five anti-88C monoclonal antibodies were tested for their

15 ability to block infection of cells by SIV, the simian immunodeficiency virus
closely related to HIV [Lehner, et aL, Nature Medicine, 2:767 (1996)].
Simian CD4"** T cells, which are normally susceptible to infection by SIV,
were incubated with the SIV mac 32HJ5 clone in the presence of the anti-88C
monoclonal antibody supernatants diluted 1:5. SIV infection was measured

20 by determining reverse transcriptase (RT) activity on day nine using the RT
detection and quantification method (Quan-T-RT assay kit, Amersham, .
Arlington Heights, IL). Four of the antibodies were able to block SIV
infection: antibody 227K blocked by 53 % , 227M by 59 % , 227N by 47 % and
227P by 81%. Antibody 227R did not block SIV infection.

25 The five monoclonal antibodies raised against human 88C

amino-terminal peptide were also tested for reactivity against macaque 88C
(SEQTlD-'KO'iK^ (which has two amino acid differences from human 88C
within the amino-terminal peptide region). # The coding regions of human 88C
and macaque 88C were cloned into the expression vector pcDNA3

30 (Invitrogen). These expression plasmids were used to transfect COS cells
using DEAE. The empty vector was used as a negative control. Three days
after transfection, cells were harvested and incubated with the five anti-88C

WO 97/22698 PCT/US96/20759

-40-

monoclonal antibodies and prepared for FACS. The results showed that four
of the five antibodies (227K, 227M, 227N, 227P) recognized macaque 88 C
while one (227R) did not. All five antibodies recognized the transfected
human 88C, and none cross-reacted with cells transfected with vector alone.

5 Example 9

Additional methods may be used to identify ligands and
modulators of the chemokine receptors of the invention.

In one embodiment, the invention comprehends a direct assay
for ligands. Detectably labeled test compounds are exposed to membrane

10 preparations presenting chemokine receptors in a functional conformation.
For example. HEK-293 cells, or tissue culture cells, are transfected with an
expression vehicle encoding a chemokine receptor. A membrane preparation
is then made from the transfected cells expressing the chemokine receptor.
The membrane preparation is exposed to 125 I-labeled test compounds (e.g. ,

15 chemokines) and incubated under suitable conditions (e.g. , 10 minutes at
37 C). The membranes, with any bound test compounds, are then collected
on a filter by vacuum filtration and washed to remove .unbound test
compounds. The radioactivity associated with the bound test compound is
then quantitated by subjecting the filters to liquid scintillation

20 spectrophotometry; The specificity of test compound binding may be
confirmed by repeating the assay in the presence of increasing quantities of
unlabeled test compound and noting the level of competition for binding to the
receptor. These binding assays can also identify modulators of chemokine
receptor binding. The previously described binding assay may be performed

25 with the following modifications. In addition to detectably labeled test
. compound, a potential modulator is exposed to the membrane preparation. An
increased level of membrane-associated label indicates the potential modulator
is an activator; a decreased level of membrane-associated label indicates the
potential modulator is an inhibitor of chemokine receptor binding.

30 In another embodiment, the invention comprehends indirect

assays for identifying receptor ligands that exploit the coupling of chemokine

WO 97/22698 PCT/US 96/20759

- 41 -

receptors to G proteins. As reviewed in Under et aL, Sci. Am., 267:56-65
(1992), during signal transduction, an activated receptor interacts with a G
protein, in turn activating the G protein. The G protein is activated by
exchanging GDP for GTP. Subsequent hydrolysis of the G protein-bound
5 GTP deactivates the G protein. One assay for G protein activity therefore,
monitors the release of 32 Pj from [y- 32 P]-GTP. For example, approximately
5 x 10 7 HEK-293 cells harboring plasmids of the invention are grown in
MEM + 10% FCS. The growth medium is supplemented with 5 mCi/ml
[ 32 P]-sodium phosphate for 2 hours to uniformly label nucleotide pools. The

10 cells are subsequently washed in a low-phosphate isotonic buffer. One aliquot
of washed cells is then exposed to a test compound while a second aliquot of
cells is treated similarly, but without exposure to the test compound.
Following an incubation period (e.g., 10 minutes), cells are pelleted, lysed
and nucleotide compounds fractionated using thin layer chromatography

15 developed with 1 M LiCl. Labeled GTP and GDP are identified by co-
developing known standards. The labeled GTP and GDP are then quantitated
by autoradiographic techniques that are standard in the art. Relatively high
levels of 32 P-labeled GDP identify test compounds as ligands. This type of
GTP hydrolysis assay is also useful for the identification of modulators of

20 - chemokine receptor binding. The aforementioned assay is performed in the
presence of a potential modulator. An intensified signal resulting from a _
relative increase in GTP hydrolysis, producing 32 P-labeIed GDP, indicates a
relative increase in receptor activity. The intensified signal therefore identifies
the potential modulator as an activator. Conversely, a diminished relative

25 signal for 32 P-labeled GDP, indicative of decreased receptor activity, identifies
the potential modulator as an inhibitor of chemokine receptor binding.

The activities of G protein effector molecules (e.g., adenylyl
cyclase, phospholipase C, ion channels, and phosphodiesterases) are also
amenable to assay. Assays for the activities of these effector molecules have

30 been previously described. For example, adenylyl cyclase, which catalyzes
the synthesis of cyclic adenosine monophosphate (cAMP), is activated by G
proteins. Therefore, ligand binding to a chemokine receptor that activates a

PCT/US96/20759

WO 97/22698

- 42 -

G protein, which in turn activates adenylyl cyclase, can be detected by
monitoring cAMP levels in a recombinant host cell of the invention.
Implementing appropriate controls understood in the art. an elevated level of
intracellular cAMP can be attributed to a ligand-induced increase in receptor
5 activity, thereby identifying a ligand. Again using controls understood in the
art, a relative reduction in the concentration of cAMP would indirectly
identify an inhibitor of receptor activity. The concentration of cAMP can be
measured by a commercial enzyme immunoassay. For example, the BioTrak
Kit provides reagents for a competitive immunoassay. (Amersham, Inc.,
10 Arlington Heights, IL). Using this kit according to the manufacturer's .
recommendations, a reaction is designed that involves competing unlabeled
cAMP with cAMP conjugated to horseradish peroxidase. The unlabeled
cAMP may be obtained, for example, from activated cells expressing the
chemokine receptors of the invention. The two compounds compete for
15 binding to an immobilized anti-cAMP antibody. After the competition
. reaction, the immobilized horseradish peroxidase-cAMP conjugate is
quantitated by enzyme assay using a tetramethylbenzidine/H 2 0 2 single-pot
substrate with detection of colored reaction products occurring at 450 nm .
The results provide a basis for calculating the level of unlabeled cAMP, using
20 techniques that are standard in the art. In addition to identifying ligands
binding to chemokine receptors, the cAMP assay can also, be used to identify
modulators of chemokine receptor binding. Using recombinant host cells of
the invention, the assay is performed as previously described, with the
addition of a potential modulator of chemokine receptor activity. By using
25 controls that are understood in the art, a relative increase or decrease in
intracellular cAMP levels reflects the activation or inhibition of adenylyl
cyclase activity. The level of adenylyl cyclase activity, in turn, reflects the
relative activity of the chemokine receptor of interest. A relatively elevated
level of chemokine receptor activity identifies an activator; a relatively
30 reduced level of receptor activity identifies an inhibitor of chemokine receptor
activity.

WO 97/22698 PCT/US96/20759

- 43 -

While the present invention has been described in terms of
specific embodiments, it is understood that variations and modifications will
occur to those skilled in the art. Accordingly, only such limitations as appear
in the appended claims should be placed on the invention.

WO 97/22698

PCT/US96/20759

- 44 -

SEQUENCE LISTING

{1) GENERAL INFORMATION :

(i) APPLICANT: I COS Corporation.

22021 20th Avenue S.E.
Bothell, WA 9 8201

(ii) TITLE OF INVENTION: Chemokiiie Receptor Materials and Methods
(iii) NUMBER OF SEQUENCES: 20

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: .Marshall, O'.Toole, Gerstein,' Murray & Borion

(B) STREET: 6300 Sears Tower, 233 S. Wacker Drive

(D) STATE: Illinois'

(E) COUNTRY: USA
' (F) ZIP : 60606

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA: . .

■ (A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY /AGENT INFORMATION: '.

(A) NAME: Noland, Greta E.

<B) REGISTRATION NUMBER: 35,302

(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 312-474-6300
(Bi TELEFAX :' 312-474-0448

(2) INFORMATION FOR SEQ ID NO : 1 : '

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 83 base pairs
• (B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE :

(A) NAME /KEY: CDS

(B) LOCATION: 55.. 1110

(ix) FEATURE :

(A) NAME /KEY : misc_feature

(D) OTHER INFORMATION: /= "88C polynucleotide and amino acid

sequences"/ .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

AGAAGAGCTG AGACATCCGT TCCCCTACAA GAAACTCTCC CCGGGTGGAA CAAG ATG 57

Met

* 1

WO 97/22698

PCT/US 96/20759

- 45

150

230

245

105'

GAT TAT CAA GTG TCA ACT CGA ATG TAT GAC ATC AAT TAT TAT ACA TCG
Asp Tyr Gin Val Ser Ser Pro He Tyr Asp- He Asn Tyr Tyr Thr Ser

GAG CCC TGC CAA AAA ATC AAT GTG AAG CAA ATC GCA GCC CGC CTC CTG 153
G?u Pro Cys Gin LyJ lie Asn Val . Lys Gin He Ala Ala Arg Leu Leu
20 25 30

CCT CCG CTC TAG TCA CTG GTG TTC ATC TTT GGT TTT GTG GGC AAC ATG 2 01

So ?ro leu Syr Ser Leu Val Phe lie Phe Gly Phe Val Giy Asn Met

35 40 45

CG GTG ATC CTC ATC CTG ATA AAC TGC AAA AGG CTG AAG AGC ATG ACT 249

leu vll S Leu lie Leu lie Asn Cys Lys Arg Leu Lys Ser Met Thr ,
50 — 55 ■ .- . . 60 55

GAC ATC TAC CTG CTC AAC CTG GCC ATC TCT GAC CTG TTT TTC CTT CTT 297
■Asp lie Tyr Leu Leu Asn Leu Ala lie Ser Asp Leu Phe Phe Leu Leu
^ J 75 80

«CT GTC CCC TTC TGG GCT CAC TAT GCT GCC GCC GAG TGG GAC TTT GGA 345
Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln. Trp Asp Phe Gly -

85 90 ■ ■ 95 .

AAT ACA ATG TGT CAA CTC TTG ACA GGG CTC TAT TTT ATA GGC TTC TTC 393
Asn Thr Met Cys Gin Leu Leu Thr Gly Leu Tyr Phe He Gly Phe Phe
100 - 105 ' .110

TCT GGA ATC TTC TTC ATC ATC CTC CTG ACA ATC GAT AGG TAC CTG GCT 441
Se- Gly He Phe Phe He lie Leu Leu Thr. He Asp Arg Tyr Leu Ala
1I5 120 125 •

GTC GTC CAT GCT GTG TTT GCT TTA AAA GCC AGG ACG GTC ACC TTT GGG 4 89

Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr. Phe Gly
130. 135 140 , 145

. GTG GTG ACA AGT GTG ATC ACT TGG GTG GTG GCT GTG. TTT GCG TCT CTC ■ 537

Val Val Thr Ser Val He Thr Trp. Val Val Ala Val Phe Ala Ser Leu

■ - -- 155 I 60

C"A GGA ATC ATC TTT ACC AGA TCT CAA AAA GAA GGT CTT CAT TAC ACC " 585
=ro Gly He He Phe Thr Arg Ser -Gin Lys Glu Gly Leu His Tyr Thr
165 . 170 175

TGC AGC TCT CAT TTT CCA TAC AGT CAG TAT CAA TTC TGG AAG AAT TTC 633
Cys Ser Ser His Phe Pro Tyr Ser Gin Tyr Gin Phe Trp Lys Asn Phe
180 185 .190

CAG ACA TTA AAG ATA GTC ATC TTG GGG CTG GTC CTG CCG CTG CTT GTC 681
Gin Thr Leu Lys lie Val lie Leu Gly Leu Val Leu Pro Leu Leu Val
195 200 205

ATG GTC ATC TGC TAC TCG GGA ATC CTA AAA ACT CTG CTT CGG TGT CGA • 729

Met Val lie Cys Tyr Ser
210 215

ATG GTG ATC TGC TAC TCG GGA ATC CTA AAA.ALT uxv* ^± ^v«*

Met Val lie Cys Tyr Ser Gly lie Leu Lys Thr Leu Leu Arg Cys Arg
210 215 220 225

AAT GAG AAG AAG AGG CAC AGG GCT GTG AGG CTT ATC TTC ACC ATC ATG 777
Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu lie Phe Thr lie Met

235 240

ATT GTT TAT TTT CTC TTC TGG GCT CCC TAC AAC ATT GTC CTT CTC CTG 825
He Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn lie Val Leu Leu Leu

250 255

AAC ACC TTC CAG GAA TTC TTT GGC CTG AAT AAT TGC AGT AGC TCT AAC 8 73

Asn Thr Phe Gin Glu Phe Phe Gly Leu Asn Asn Cys Ser Ser -Ser Asn
260 265 270

WO 97/22698

PCT/US 96/20759

-46-

AGG TTG GAC CAA GCT ATG CAG GTG ACA GAG ACT CTT GGG ATG ACG CAC 9 21

Arg Leu Asp Gin Ala Met Gin Val Thr Glu thr Leu Gly Met Thr His
275 280 . 285

TGC TGC ATC AAC CCC ATC ATC TAT GCC TTT GTC GGG GAG AAG TTC AGA 9 69

Cys Cys lie Asn Pro lie lie Tyr Ala Phe Val Gly Glu Lys Phe Arg
290 295 1 300 305

AAC TAG CTC TTA GTC TTC TTC CAA AAG CAC ATT GCG AAA CGC TTC TGC 1017
Asn Tyr. Leu Leu Val Phe Phe Gin Lys His lie Ala Lys Arg, Phe Cys
310 315 - 320

AAA TGC TGT TCT ATT TTC CAG . CAA GAG GCT CCC GAG CGA GCA AGC TCA 1065
Lys Cys Cys Ser He Phe Gin Gin, Glu Ala Pro Glu Arg Ala Ser Ser
325 330 . 335

GTT TAC ACC CGA* TCC ACT GGG GAG CAG GAA ATA TCT GTG GGC TTG 1110
Val Tyr Thr Arg Ser Thr Gly Glu Gin Glu He Ser Val 1 Gly Leu

340

345

350

TGACACGGAC

TCAAGTGGGC

TGGTGAC C CA

GTCAGAGTTG

TGCACATGGC

TTAGTTTTCA

1170

TACACAGCCT

GGGCTGGGGG

TGGGGTGGGA

GAGGTCTTTT

TTAAAAGGAA

GTTACTGTTA .

1230

TAGAGGG'TCT

AAGATTCATC

CATTTATTTG

GCATCTGTTT

AAAG TAGATT

AGATCTTTTA

1290

AG C C CATC AA

TTATAGAAAG

CCAAATCAAA

ATATGTTGAT

GAAAAATAGC

AAC CTTTTTA

1350

TCTCCCCTTC

ACATG CAT CA

AGTTATTGAC

AAACTCTCCC

TTCACTCCGA

AAGTTC CTTA

1410

TGTATATTTA

AAAGAAAGCC

TCAGAGAATT

GCTGATTCTT

GAGTTTAGTG

ATCTGAACAG

1470

AAATACCAAA

ATTATTTCAG

AAATG TACAA

CTTTTTACCT

AGTACAAGGC

AACATATAGG

1530

TTGTAAATGT

GTTTAAAACA

GGTCTTTGTC

TTGCTATGGG

GAGAAAAGAC

ATGAATATGA ■

1590

TTAGTAAAGA

AATGACACTT

TTCATGTGTG

ATTTCCCCTC

CAAGGTATGG

TTAATAAGTT

1650

TCACTGACTT

AGAAC CAGGC

GAGAGACTTG

TGGCCTGGGA

GAGCTGGGGA

AGCTTCTTAA

1710

ATGAGAAGGA

ATTTGAGTTG

GATCATCTAT

TGCTGGCAAA

GACAGAAGCC

TCACTGCAAG

1770

CACTGCATGG

GCAAGCTTGG

CTGTAG AAG G

AGACAGAGCT

GGTTGGGAAG

ACATGGGGAG

1830

GAAGGACAAG

GCTAGATCAT

GAAGAACCTT

GACGGCATTG

CTCCGTCTAA

GTCATGAGCT

1890

GAGCAGGGAG

ATCCTGGTTG

GTGTTG CAGA

AGGTTTACTC

TGTGGCCAAA

GGAGGGTCAG

1950

GAAGGATGAG

CATTTAGGGC

AAGGAGACCA

CCAACAGCCC

TCAGGTCAGG

GTGAGGATGG

2010

CCTCTGCTAA

GCTCAAGGCG

TGAGGATGGG

AAGGAGGGAG

GTATTCGTAA

GGATGGGAAG

2070

GAGGGAGGTA

TTCGTGCAGC

ATATGAGGAT

GCAGAGTCAG

CAGAACTGGG

GTGGATTTGG

2130

TTTGGAAGTG

AGGGTCAGAG

AGGAGTCAGA

GAGAATCCCT

AGTCTTCAAG

CAGATTGGAG

2190

AAACCCTTGA

AAAGACATCA

AGCACAGAAG

GAGGAGGAGG

AGGTTTAGGT

CAAGAAGAAG

2250

ATG GATTGGT

GTAAAAGGAT

GGGTCTGGTT

TGCAGAGCTT

GAACACAGTC

TCACCCAGAC

2310

TCCAGGCTGT

CTTTCACTGA

ATG CTTCTGA

CTT CAT AG AT

TTCCTTCCCA

TCCCAGCTGA

2370

AATACTGAGG

GGTCTCCAGG

AGGAGACTAG

ATTTATGAAT

ACACGAGGTA

TGAGGTCTAG

2430

GAACATACTT

CAGCTCACAC

ATGAGATCTA

GGTGAGGATT

GATTACCTAG

TAGTCATTTC

2490

ATGGGTTGTT

GGGAGGATTC

TATGAGGCAA

CCACAGGCAG

CATTTAGCAC

ATACTACACA

2550

WO 97/22698 PCT/US96/20759

- 47 -

TTC AATAAG C

ATCAAACTCT

TAGTTACTCA

TTCAGGGATA

GCACTGAGCA

AAG CATTG AG

2610

CAAAGGGGTC

CCATATAGGT

GAGGGAAGCC

TGAAAAACTA

AGATGCTGCC

TGCCCAGTGC

2670

ACACAAGTGT

AGGTATCATT

TTCTGCATTT

AACCGTCAAT

AGGCAAAGGG

GGGAAGGGAC

2730

ATATTCATTT

GGAAATAAGC

TGCCTTGAGG

CTTAAAACCC

ACAAAAGTAC

AATTTAC CAG

2790

CCTCCGTATT

TCAGACTGAA

TGGGGGTGGG

GGGGGCGCCT

TAGGTACTTA

TTC CAGATG C

2850

CTTCTCCAGA

CAAACCAGAA

GCAACAGAAA

AAATCGTCTC

TCCCTCCCTT

TGAAATGAAT .

2910

ATACCCCTTA

GTGTTTGGGT

ATATTCATTT

CAAAGGGAGA

GAGAGAGGTT

TTTTT CTGTT

• 2970,

CTTTCTCATA

TGATTGTGCA

CATACTTGAG

ACTGTTTTGA

ATTTGGGGGA

TGGCTAAAAC

3030

CATCATAGTA

CAGGTAAGGT

GAGGGAATAG

TAAGTGGTGA

GAACTACTCA

GGGAATGAAG

3090

GTGTCAGAAT

AATAAGAGGT

GCTACTGACT

TTCTCAGCCT

CTGAATATGA

ACGGTGAGCA

3150

TTGTGGCTGT

CAG CAGGAAG

CAACGAAGGG

AAATGTCTTT

CCTTTTGCTC

TTAAGTTGTG

3210

GAGAGTGCAA

CAGTAGCATA

GGACCCTACC

CTCTGGGCCA

AGTCAAAGAC

ATTCTGACAT

3270

CTTAGTATTT

GCATATTCTT

ATGTATGTGA

AAGTTACAAA

TTGCTTGAAA

GAAAATATGC

3330

ATCTAATAAA

AAACAC C TTC

TAAAATAAAA

AAAAAAAAAA

AAA

3383

(2) INFORMATION FOR SEQ ID NO : 2 :

<i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3 52 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

. (ix) FEATURE:

(A) NAME/KEY: misc_f eature
" (D) OTHER INFORMATION: /= "88C amino acid sequence"

(xiJ SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Asp TVr Gin Val Ser Ser Pro lie Tyr Asp lie Asn Tyr Tyr "Thr
1 - 5 10 15

Ser Glu Pro Cys Gin Lys lie Asn Val Lys Gin lie Ala Ala Arg Leu
20 25 30

Leu Pro Pro Leu Tyr Ser Leu Val Phe lie Phe Gly Phe Val Gly. Asn
35 40 45

Met Leu Val He Leu He Leu lie Asn Cys Lys Arg . Leu Lys Ser Met
5 0 55 - 60

Thr Asp He Tyr Leu Leu Asn Leu Ala He Ser Asp Leu Phe Phe Leu
65 70- 75 80

Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gin Trp Asp Phe
85 90 95

Gly Asn Thr Met Cys Gin Leu Leu Thr Gly Leu Tyr. Phe He Gly Phe
100 105 110

Phe Ser Gly He Phe Phe He He Leu Leu Thr He Asp Arg Tyr Leu
115 120 125

WO 97/22698 PCT/US9 6/20759

- 48 -

Ala Val Val His Ala Val Phe Ala Leu Lys Ala^Arg Thr'Val Thr Phe
130 135 140

Gly Val Val Thr Ser Val lie Thr Trp Val Val Ala Val Phe Ala Ser
145 150 155 160

Leu Pro Gly He He Phe Thr Arg Ser Gin Lys Glu Gly Leu His Tyr
165 170 175

Thr* Cys Ser Ser His Phe Pro Tyr Ser Gin Tyr Gin Phe Trp Lys Asn
180 185 190

Phe Gin Thr Leu Lys He Val He Leu Gly Leu Val Leu Pro Leu Leu
195 200 205

Val Met Val lie Cys Tyr Ser Gly He Leu Lys Thr Leu Leu Arg Cys
210 215 - 220

Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu He Phe Thr He
225 230 .235 240

Met He Val' Tyr' Phe Leu Phe Trp Ala Pro Tyr Asn He Val Leu Leu
245 250 255

Leu Asn Thr Phe Gin Glu Phe Phe Gly Leu Asn' Asn Cys Ser Ser Ser
260 265 270

Asn Arg Leu Asp Gin Ala Met Gin Val Thr Glu Thr Leu Gly Met Thr
275 280 * 285

His Cys Cys He Asn Pro He He Tyr Ala Phe Val Gly Glu Lys Phe
230 ' 295 300

Arg Asn Tyr Leu Leu Val Phe Phe Gin Lys His He Ala Lys Arg Phe
305 310 315 . 320

Cys Lys Cys Cys Ser He Phe Gin Gin Glu Ala Pro Glu Arg Ala Ser
325 330 335

Ser Val Tyr Thr Arg Ser Thr Gly Glu Gin Glu He Ser Val Gly Leu
340 345 350

(2) INFORMATION FOR SEQ ID NO : 3 :

(ij SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1915 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY : linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION : .362. . 1426

(ix) FEATURE:

(A) NAME /KEY : misc_f eature

(D) OTHER INFORMATION: /= " 88-2B polynucleotide and amino acid

sequences"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
ATAATAATGA TTATTATATT GTTATCATTA TCTAGCCTGT TTTTTCCTGT TTTGTATTTC 60

TTCCTTTAAA TGCTTTCAGA AATCTGTATC CCCATTCTTC ACCACCACCC CACAACATTT 12 0

WO 97/22698

PCT/LIS96/20759

- 49 -

CTGCTTCTTT TCCCATGCCG GGTCATGCTA ACTTTGAAAG CTTCAGCTCT TTCCTTCCTC ^ 18 0

AATCCTTTTC CTGGCACCTC TGATATGCCT TTTGAAATTC ATGTTAAAGA ATCCCTAGGC 24 0

- TGCTATCACA TGTGGCATCT TTGTTGAGTA CATGAATAAA TCAACTGGTG TGTTTTACGA ' 3 00

AGGATGATTA TGCTTCATTG TGGGATTGTA TTTTTCTTCT TCTATCACAG GGAGAAGTGA 3 60

A ATG ACA ACC TCA CTA GAT ACA GTT GAG ACC TTT GGT ACC ACA TCC 4 06

Met Thr Thr Ser Leu Asp Thr Val Glu Thr Phe Gly Thr Thr Ser
1 5 10 - 15

TAC TAT GAT GAC GTG GGC CTG CTC TGT GAA AAA GCT GAT ACC AGA GGA 4 54

Tyr Tyr Asp Asp Val Gly Leu Leu Cys Glu Lys Ala Asp Thr, Arg Ala
20 25 ' 30

CTG ATG GCC CAG TTT GTG CCC CCG'CTG TAG TCC CTG GTG TTC ACT GTG 5 02

Leu Met Ala Gin Phe Val Pro Pro Leu Tyr Ser Leu Val Phe Thr Val
35 40 ,45

GGC CTC TTG GGC AAT GTG GTG GTG GTG ATG ATC CTC ATA AAA TAC AGG 55 0

Gly Leu Leu Gly Asn Val Val Val Val Met He- Leu lie -Lys Tyr Arg
50 55 . 60

AGG CTC CGA ATT ATG ACC AAC ATC TAC CTG CTC AAC CTG GCC ATT TCG 59 8

Arg Leu Arg lie Met Thr Asn lie Tyr Leu Leu Asn Leu Ala . lie Ser
65 70 75 .

GAC CTG CTC TTC CTC GTC ACC CTT CCA TTC TGG ATC CAC TAT GTC AGG 64 6

Asp Leu Leu Phe Leu Val Thr Leu Pro Phe Trp lie His Tyr Val Arg
80 ' * 85 90 95,

GGG CAT AAC TGG GTT TTT GGC CAT GGC ATG TGT AAG CTC CTC TCA GGG * 694"
Gly 'His Asn Trp Val Phe Gly His Gly Met Cys Lys Leu Leu Ser Gly .

100 ' 105 ' 110 t

TTT TAT CAC ACA GGC TTG TAC AGC GAG ATC TTT TTC ATA. ATC CTG CTG - 74 2

Phe Tyr His Thr Gly Leu Tyr Ser Glu lie Phe Phe lie lie Leu Leu
115 120 . . 125

ACA ATC GAC AGG TAC CTG GCC ATT GTC CAT GCT GTG TTT GCC CTT CGA . . 79 0

Thr He Asp Arg Tyr Leu Ala He Val His- Ala Val Phe Ala Leu Arg -
130 135 . . 140' ' •

GCC CGG ACT GTC ACT TTT GGT GTC . ATC ACC AGC ATC GTC ACC TGG GGC 83 8

Ala Arg Thr Val Thr Phe Gly Val lie Thr Ser lie Val Thr 'Trp Gly
145 150 ' 155

CTG GCA GTG CTA GCA GCT CTT CCT GAA TTT ATC TTC TAT GAG ACT GAA' 8 86

Leu Ala Val Leu Ala Ala Leu Pro Glu Phe He Phe Tyr Glu Thr Glu
160 165 170 175

GAG TTG TTT GAA GAG ACT CTT TGC AGT GCT CTT TAC CCA GAG GAT ACA 9 34

Glu Leu Phe Glu Glu Thr Leu Cys Ser Ala Leu Tyr Pro Glu Asp Thr
180 185 190

GTA TAT AGC TGG AGG CAT TTC CAC ACT CTG AGA ATG ACC ATC TTC TGT 9 82

Val Tyr Ser Trp Arg His Phe His Thr Leu Arg Met Thr He Phe Cys
195 * . 200 205

CTC GTT CTC CCT CTG CTC GTT ATG GCC ATC TGC TAC ACA GGA ATC ATC 10 3 0

Leu Val Leu Pro Leu Leu Val Met Ala He Cys Tyr Thr Gly lie He
210 215 220

AAA ACG CTG CTG AGG TGC CCC AGT AAA AAA AAG TAC AAG GCC ATC CGG 107 8

Lys Thr Leu Leu Arg Cys Pro Ser Lys Lys Lys Tyr Lys Ala He Arg
225 230 235

WO 97/22698

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PCT/US9 6/20759

CTC ATT TTT GTC ATC ATG GCG GTG TTT TTC ATT TTC TGG ACA CCC TAC 1126
Leu lie Phe Val lie Met Ala Val Phe Phe lie Phe Trp Thr Pro Tyr
240 245 ' 250 . 255

AAT GTG GGT ATC CTT CTC TCT TCC TAT CAA TCC ATC TTA TTT GGA AAT 1174
Asn Val Ala lie Leu Leu Ser Ser Tyr Gin Ser lie Leu Phe Gly Asn .

260 . 265 . 270

GAC TGT GAG CGG AGC AAG CAT CTG GAC CTG GTC ATG CTG GTG ACA GAG 1222
Asp Cys Glu Arg Ser Lys His Leu Asp Leu Val Met Leu Val Thr Glu
. 275 280 285

GTG ATC GCC TAC TCC CAC TGC TGC ATG ■ AAC CCG GTG ATC TAC GCC TTT 12 70

Val lie Ala Tyr Ser His Cys Cys Met Asn Pro Val lie Tyr Ala Phe .
29 0 295 3 00

GTT GGA GAG AGG TTC . CGG AAG TAC CTG CGC' CAC TTC TTC CAC AGG CAC 1318
Val Gly Glu Arg 'Phe Arg Lys Tyr Leu Arg His Phe, Phe His Arg His
305 310 315

TTG CTC ATG CAC CTG ' GGC AGA TAC ATC CCA TTC CTT CCT AGT GAG AAG 13 6 6

Leu Leu Met His Leu Gly Arg Tyr lie Pro Phe Leu Pro Ser Glu Lys
320 . 325 330 * 335

CTG GAA AGA ACC AGC TCT GTC TCT CCA TCC ACA GCA GAG CCG GAA CTC '1414
Leu Glu Arg Thr Ser Ser Val Ser Pro Ser Thr Ala Glu Pro Glu Leu
340 345 350

TCT ATT GTG TTT TAGGTCAGAT G C AG AAAATT GCCTAAAGAG GAAGGACCAA 14 6 6
Ser Tie Val Phe
355

GGAGATGAAG CAAACACATT AAGCCTTCCA CACTCACCTC TAAAACAGTC CTTCAAACTT 152 6

CCAGTGCAAC ACTGAAGCTC TTG AAG A CAC TGAAATATAC ACACAG CAGT AGCAGTAG AT 158 6
GCATGTACCC TAAGGTCATT ACCACAGGCC AGGGGCTGGG CAGCGTACTC ATCATCAACC . 164 6

CTAAAAAGCA GAGCTTTGCT TCTCTCTCTA AAATG AGTTA CCTACATTTT AATGCACCTG 17 06

AATGTTAGAT AGTTACTATA TGCCGCTACA AAAAGGTAAA ACTTTTTATA TTTTATACAT 176 6

TAACTTCAGC CAG CTATTG A TATAAATAAA ACATTTTCAC ACAATACAAT AAGTTAACTA 182 6
TTTTATTTTC TAATGTGCCT AGTTCTTTCC CTGCTTAATG AAAAGCTTGT TTTTTCAGTG • 1886

TGAATAAATA ATCGTAAGCA ACAAAAAAA 1915
(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 35 5 amino acids

(B) TYPE: amino. acid
(D) TOPOLOGY :* linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME /KEY : miscjeature

(D) OTHER INFORMATION: /= "88-2B amino acid sequence"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Thr Thr Ser Leu Asp Thr Val Glu Thr Phe Gly Thr Thr Ser Tyr
1 5 10 15

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Tvr Asp Asp Val Gly Leu Leu Cys Glu Lys Ala Asp Thr Arg Ala Leu
20 25 30

Met Ala Gin Phe Val Pro Pro Leu Tyr Ser Leu Val Phe Thr Val Gly
35 40 45

Leu Leu Gly Asn Val Val Val Val Met lie Leu lie Lys Tyr Arg Arg
50 55 60

Leu Arg He Met Thr Asn He Tyr Leu Leu Asn Leu Ala lie Ser Asp
65 70 75 80

Leu Leu Phe Leu Val Thr Leu Pro Phe Trp He His Tyr Val Arg Gly
85 90 -95 v

His Asn Trp Val Phe Gly His Gly Met Cys Lys Leu Leu Ser Gly Phe
100- 105 110.

Tyr His Thr Gly Leu Tyr Ser Glu He Phe Phe 'He He Leu Leu Thr
115 .120 • 125

He Asp Arg Tyr Leu Ala He Val His Ala Val Phe Ala Leu Arg Ala
130 135 140

Arg Thr Val Thr Phe Gly Val He Thr Ser He Val Thr Trp Gly Leu
145 150 155 160

Ala Val Leu Ala" Ala Leu Pro Glu Phe He Phe Tyr Glu Thr Glu Glu
165 170 175

Leu Phe Glu Glu Thr Leu Cys Ser Ala Leu Tyr, Pro Glu Asp Thr Val
180 185 190

Tyr Ser Trp Arg His Phe His Thr* Leu Arg Met Thr He Phe Cys Leu
195 200 ,205

Val Leu Pro Leu Leu Val Met Ala He. Cys Tyr Thr Gly He He Lys
210 215 220

Thr Leu Leu Arg' Cys Pro Ser Lys Lys Lys Tyr Lys Ala He Arg Leu
225 230 235 ■ 240

He Phe Val He Met Ala Val Phe Phe He Phe Trp Thr Pro Tyr Asn
245 250 255

Val Ala He Leu Leu Ser Ser Tyr Gin Ser lie Leu Phe Gly Asn Asp
260 265 270

Cys Glu Arg Ser Lys His Leu Asp Leu Val Met Leu Val Thr Glu Val
275 • 280 285

lie Ala Tyr Ser His Cys Cys Met Asn Pro Val He Tyr Ala Phe Val
290 295 * 300

Gly Glu Arg Phe Arg Lys Tyr Leu Arg His Phe Phe His Arg His Leu
305 310 315 320

Leu Met His Leu Gly Arg Tyr He Pro Phe Leu Pro Ser Glu Lys Leu
325 330 335

Glu Arg Thr Ser Ser Val Ser Pro Ser Thr Ala Glu Pro Glu Leu Ser
340 345 350

■He Val Phe

- 355

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(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: ; .DNA

(ix) FEATURE :

(A) NAME /KEY : misc_f eature

(D) OTHER INFORMATION : /= "V2 8degf2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

GACGGATCCA TYGAYAGRTA CCTGGCYATY GTCC .

(2) INFORMATION FOR SEQ ID NO : 6 :

£i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3 2 base pairs

(B) TYPE: nucleic acid

(D) ' TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME /KEY : mi sc_f eature

(D) OTHER- INFORMATION: /= "V2 8degr2"

(xi) SEQUENCE. DESCRIPTION: SEQ ID NO : 6 :

GCTAAGCTTT TRTAGGGDGT CCAYAAGAGY AA

(2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE: *

(A) NAME/KEY: misc_feature

<D) OTHER INFORMATION: /= "88c-r4 "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :

GATAAGCCTC ACAGCCCTGT G

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2 8 base pairs

(B) TYPE: nucleic acid

( D ) TOPOLOGY : 1 inear

(ii) MOLECULE TYPE: DNA

(ix)

FEATURE :
(A) NAME /KEY : misc_feature
(D) OTHER INFORMATION: /= "88c-rlb n

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
GCTAAGCTTG ATGACTATCT TTAATGTC
(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs .

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

' (ii) MOLECULE TYPE: DNA

( ix) FEATURE :

(A) NAME/KEY :■ mis cofeature

(D) OTHER INFORMATION: /= "88-2B-3"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 : ,

CCCTCTAGAC TAAAACACAA TAGAGAG ."

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid .
■(C)' .STRANDEDNESS : single
(D) TOPOLOGY : linear

. (ii) MOLECULE TYPE : ; DNA

(ix) FEATURE:

(A) NAME /KEY : misc^f eature

(D) OTHER INFORMATION: /= "88-2B-5 n

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GCTAAGCTTA T CACAGGG AG AAGTGAAATG

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2 0 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

•' (ii) MOLECULE TYPE: DNA '

(ix) FEATURE:

(A) NAME /KEY : misc_f eature

(D) OTHER INFORMATION: /= "88-2B-fl"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

AGTGCTAGCA GCTCTTCCTG

(2) INFORMATION FOR SEQ ID NO: 12:

(i). SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2 0 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY : linear

(ii) MOLECULE TYPE: DNA

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(ix) FEATURE:

(A) NAME / KEY : misc_f eature

(D) OTHER INFORMATION: /= n 88-2B-rl M

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CAGCAGCGTT TTGATGATTC 2 0

f2) INFORMATION .FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS: *
(A) LENGTH: 19 base pairs
' {B) TYPE: nucleic acid

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME /KEY : misc_f eature

(D) OTHER INFORMATION: /= n 88C-fl"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

TGTGTTTGCT TTAAAAGCC 19

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:
, (A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

( A) NAME /KEY : mis c_f eature

(D) OTHER INFORMATION: /= n 8 8C-r3 M

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

TAAGCCTCAC AGCCCTG * '17

(2) INFORMATION FOR SEQ . ID NO: 15: •

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2 8 base pairs
• (B) TYPE: nucleic acid

( D ) TOPOLOGY : 1 i ne ar

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME /KEY : misc_f eature

(D) OTHER INFORMATION: /= " CCCKR1 (2 ) - 5« Primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CGTAAGCTTA GAGAAGCCGG GATGGGAA 2 8

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

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(ii) MOLECULE TYPE: DNA '

<iv) ANTI- SENSE : YES

<ix) FEATURE : '
(A) NAME /KEY : misc_f earure

(D) OTHER INFORMATION: /= "CCCKR-3" Primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GCCTCTAGAG TCAGAG AC CA GCAGA . 25

(2) INFORMATION FOR SEQ ID NO: 17,: .

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2 8 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear ,
(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GACAAGCTTC ACAGGGTGGA ACAAGATG - ?8

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE. TYPE : DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GTCTCTAGAC CACTTGAGTC CGTGTCA 27
(2) INFORMATION FOR SEQ ID NO: 19:

. (i) SEQUENCE CHARACTERISTICS: ■

(A) LENGTH: 1059 base pairs

(B) TYPE: nucleic acid

■ (C) STRANDEDNESS: single
(D) TOPOLOGY : • linear

(ii) MOLECULE TYPE: cDNA v *

(ix) FEATURE:

( A) NAME /KEY: CDS

(B) LOCATION: 1..1056

(xi)

SEQUENCE DESCRIPTION: SEQ ID NO : 19 :

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ATG GAC TAT CAA GTG TCA AGT CCA ' ACC TAT GAC ATC GAT TAT TAT ACA 4 8

Met Asp Tyr Gin Val Ser Ser Pro Thr Tyr Asp lie Asp Tyr Tyr Thr
1 5 10 • 15

TCG GAA CCC TGC CAA AAA ATC AAT GTG AAA CAA ATC GCA GCC . CGC CTC 9 6

Ser Glu Pro Cys Gin Lys lie Asn Val Lys Gin lie Ala Ala Arg Leu
20 .25 30

CTG CCT CCG CTC TAG TCA CTG GTG ^TTC ATC TTT GGT TTT GTG GGC AAC 14 4

Leu Pro Pro Leu Tyr Ser Leu Val Phe lie Phe Gly Phe Val Gly Asn
35 40 . 45

ATA CTG GTC GTC CTC ATC CTG ATA AAC TGC AAA AGG CTG AAA AGC ATG 19 2

lie Leu Val ' Val Leu lie Leu lie Asn Cys Lys Arg Leu Lys Ser Met
50 55 60

ACT GAC ATC TAC CTG CTC AAC CTG GCC ATC TCT GAC CTG CTT TTC CTT 24 0

Thr Asp lie Tyr Leu Leu Asn Leu Ala lie Ser Asp Leu Leu Phe Leu
65 70 75 - 80

CTT ACT GTC CCC TTC TGG GCT CAC TAT GCT GCT GCC CAG TGG GAC TTT ♦ 2 88

Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gin Trp Asp Phe
85 90 95

GGA AAT ACA ATG TGT .CAA CTC TTG ACA GGG CTC TAT TTT ATA GGC TTC 3 36

Gly Asn Thr Met Cys Gin Leu Leu Thr. Gly Leu Tyr Phe lie Gly Phe
100 105 110

TTC TCT GGA ATC TTC TTC ATC ATC "CTC CTG ACA ATC GAT AGG TAC. CTG 3 84

Phe Ser Gly lie Phe Phe lie lie Leu Leu Thr lie Asp Arg Tyr Leu
115 120 125

GCT ATC' GTC CAT GCT GTG TTT GCT TTA AAA GCC AGG ACA GTC ACC TTT 4 32

Ala lie Val His Ala Val Phe Ala Leu Lys Ala- Arg Thr Val Thr Phe
• 130 135 140

GGG GTG GTG ACA AGT GTG ATC ACT TGG GTG GTG GCT GTG TTT GCC TCT 4 80

Gly Val Val Thr Ser Val' lie Thr Trp Val Val Ala Val Phe Ala Ser
145 150 155 . 160

CTC CCA GGA ATC ATC TTT ACC AGA TCT CAG AGA GAA GGT CTT CAT TAC 52 8

Leu Pro Gly lie lie Phe Thr Arg Ser Gin Arg Glu Gly Leu His Tyr
165 170 175

ACC TGC AGC TCT CAT TTT CCA TAC AGT CAG TAT CAA TTC TGG AAG AAT 5 76

Thr Cys Ser Ser His Phe Pro Tyr Ser Gin Tyr Gin Phe Trp Lys Asn
180 185 190

TTT CAG ACA TTA AAG ATG GTC ATC TTG GGG CTG GTC CTG CCG CTG CTT 624
Phe Gin Thr Leu Lys Met Val He Leu Gly Leu Val Leu Pro Leu Leu
195 200 205

GTC ATG GTC ATC TGC TAC TCG GGA ATC CTG AAA ACT CTG CTT CGG TGT " 672
Val Met Val He Cys Tyr Ser Gly He Leu Lys Thr Leu Leu Arg Cys
210 215 220

CGA AAC' GAG AAG AAG AGG CAC AGG GCT GTG AGG CTT ATC TTC ACC ATC 720
Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu He Phe Thr He
225 230 235 240

ATG ATT GTT TAT TTT CTC TTG TGG GCT CCC TAC AAC ATT GTC CTT CTC . 7 68

Met He Val Tyr Phe Leu Leu Trp Ala Pro Tyr Asn He Val Leu Leu
245 ' 250 255

CTG AAC ACC TTC CAG GAA TTC TTT GGC CTG AAT AAT TGC AGT AGC TCT 816
Leu Asn Thr Phe Gin Glu Phe Phe Gly Leu Asn Asn Cys Ser Ser Ser
260 265 270

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AAC AGG TTG GAC CAA GCC ATG GAG GTG ACA GAG ACT CTT' GGG ATG AGA 864
Asn Arg Leu Asp Gin Ala Met Gin Val Thr Glu Thr Leu Gly Met Thr
275 280 285

CAC TGC TGC ATC AAC CCC ATC ATC TAT GCC TTT GTC GGG GAG AAG TTC 912
His Cys Cys lie Asn Pro lie lie Tyr Ala Phe Val Gly Glu Lys Phe

290 295 300 '

AGA AAC TAC CTC TTA GTC TTC TTC CAA AAG CAC ATT GCC AAA CGC TTC '9 60

Arg Asn Tyr Leu Leu Val Phe Phe Gin Lys His lie Ala Lys Arg Phe
305 ' 310 315 . 32.0

TGC AAA TGC TGT T£C ATT TTC CAG CAA GAG GCT CCC GAG CGA GCA AGT * 1008
Cys Lys Cys Cys Ser lie Phe Gin Gin Glu Ala Pro Glu Arg Ala Ser
325 330 335

TCA GTT TAC ACC CGA TCC ACT GGG GAG CAG GAA ATA TCT GTG GGC TTG .- 1056
Ser Val Tyr Thr Arg Ser Thr Gly Glu Gin Glu .lie Ser Val Gly Leu
34 0 345 350

TGA - *

(2) INFORMATION FOR SEQ ID NO : 2 0 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 352 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear .

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Met Asp Tyr Gin Val Ser Ser Pro Thr Tyr Asp lie Asp Tyr Tyr Thr
1 5 ~ 10 15

Ser Glu Pro Cys Gin Lys He Asn Val Lys Gin He Ala Ala Arg. Leu
20 : 25 30

Leu Pro Pro, Leu Tyr Ser Leu Val Phe lie Phe .Gly Phe Val Gly Asn
35 40 45

He Leu Val Val Leu He Leu He Asn Cys Lys Arg Leu Lys Ser Met
50 55 60

Thr Asp lie Tyr Leu Leu Asn Leu Ala He Ser Asp Leu Leu Phe Leu
65 70 75 80

Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gin Trp Asp Phe
85 90 95

Gly Asn Thr Met Cys Gin Leu Leu Thr Gly Leu Tyr Phe He Gly Phe
100 105 * 110

Phe Ser Gly lie Phe Phe He He. Leu Leu Thr He' Asp Arg Tyr Leu
115 120 125

Ala He Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe
130 135 140

Gly Val Val Thr Ser Val He Thr Trp Val Val Ala Val Phe Ala Ser
145 150 155 160

Leu Pro Gly He lie Phe Thr Arg Ser Gin Arg Glu Gly Leu His Tyr
165 170 175

1059

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Thr Cys Ser Ser His Phe Pro Tyr Ser Gin Tyr Gin Phe Trp Lys Asn
. 180 185 190

Phe Gin Thr Leu Lys Met Val He Leu Gly Leu Val Leu Pro Leu Leu
'195 200 205

Val Met Val He Cys Tyr Ser Gly He Leu Lys Thr Leu Leu Arg Cys
210 215 220

Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu He Phe Thr He
225 230 235 240

Met He Val Tyr Phe Leu Leu Trp Ala Pro Tyr Asn He Val Leu Leu
245 250 255

Leu Asn Thr Phe Gin Glu Phe Phe Gly Leu Asn Asn Cys Ser Ser Ser
260 265 270

Asn Arg Leu Asp Gin Ala Met Gin Val' Thr Glu Thr Leu Gly Met Thr
275 280 285

His Cys Cys He Asn Pro He He Tyr Ala Phe Val Gly Glu Lys Phe
290 295 300

Arg Asn Tyr Leu Leu Val Phe Phe Gin Lys His He Ala Lys Arg Phe
305 " 310 315 320

Cys Lvs Cys Cys Ser lie Phe Gin Gin Glu Ala Pro Glu Arg Ala Ser
325 330 335

Ser Val Tyr Thr Arg Ser Thr Gly Glu Gin Glu He Ser Val Gly Leu
340 345 350

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CLAIMS

We claim:

1 . A purified and isolated polynucleotide encoding the amino
acid sequence of chemokine receptor 88-2B set out in SEQ ID NO: 4.

2. A polynucleotide according to claim 1 wherein the
polynucleotide is DNA.

3. A polynucleotide according to claim 2 wherein the
polynucleotide is genomic DNA.

4. A polynucleotide according to claim 2 wherein the
polynucleotide is cDNA.

5. A polynucleotide according to claim 1 which is a wholly or
partially chemically synthesized DNA.

6. An RNA transcript of the polynucleotide of claim 2.

7. A cDNA according to claim 4 comprising the DNA of SEQ
ID NO:3.

8. A biologically functional DNA vector comprising a DNA
according to claim 2.

9. A vector according to claim 8 wherein said DNA is
operatively linked to a DNA expression control sequence.

10. A host cell stably transformed or transfected with a DNA
according to claim 1 in a manner allowing expression of said DNA.

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11. A method for producing an 88-2B polypeptide comprising
the steps of growing a host cell according to claim 10 in a suitable nutrient
medium and isolating said polypeptide from said cell or medium.

12. A polynucleotide encoding an 88-2B polypeptide wherein
said polynucleotide hybridizes under stringent hybridization conditions to
the polynucleotide of SEQ ID NO: 3.

13. A purified and isolated polypeptide comprising the
chemokine receptor 88-2B amino acid sequence set out in SEQ ID NO: 4.

14. An antibody product that specifically binds a polypeptide
. comprising the 88-2B amino acid sequence set out in -SEQ ID NO:4.

15. A hybridoma producing an antibody product according to
claim 14.

16. A purified and isolated polynucleotide encoding the amino
acid sequence of chemokine receptor 88C set out in SEQ ID NO:2.

17. A polynucleotide according to claim 16 wherein the
polynucleotide is DNA.

18. A polynucleotide according to claim 17 wherein the
polynucleotide is genomic DNA.

19. A polynucleotide according to claim 17 wherein the
polynucleotide is a cDNA.

20. A polynucleotide according to claim 16 which is a wholly or
partially chemically synthesized DNA.

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21 . An RNA transcript of the polynucleotide of claim 17.

22. A cDNA according to claim 19 comprising the DNA of SEQ .
ID NO: 1 .

• 23 . A biologically functional DNA vector comprising a DNA
according to claim 17.

24. A vector according to claim 23 wherein said DNA is
operatives linked to a DNA expression control sequence:

25 . A host cell stably transformed or transfected with a DNA
according to claim 16 in a manner allowing expression of said DNA,

26 A method for producing an 88C polypeptide comprising the
steps of growing a host cell according to claim 25 in a suitable nutrient
medium and isolating said polypeptide from said cell or medium.

27 A polynucleotide encoding an 88C polypeptide wherein said
polynucleotide hybridizes under stringent hybridization conditions to the
polynucleotide of SEQ ID NO: 1 .

28. A purified and isolated polypeptide comprising the
chemokine receptor 88C amino acid sequence set out in SEQ ID NO:2.

29. An antibody product that specifically binds a polypeptide
comprising the 88C amino acid sequence set out in SEQ ED NO:2.

30. A hybridoma producing an antibody product according to

claim 29.

31. Hybridoma cell line 227K.

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32. Hybridoma cell line 227M.

33. Hybridoma ceil line 227N.

34. Hybridoma cell line 227P.

35. Hybridoma cell line*227R.

36. A purified and isolated polynucleotide encoding the amino
acid sequence of macqaqiie chemokine receptor 88C set out in SEQ ID
NO: 20.

37. A polynucleotide according to claim 36 wherein the
polynucleotide is DNA.

38. A polynucleotide according to claim 37 wherein the
polynucleotide is genomic DNA.

39. A polynucleotide according to claim 37 wherein the
polynucleotide is a cDNA.

40. A polynucleotide according to claim 36 which is a wholly or
partially chemically synthesized DNA.

41. An RNA transcript of the polynucleotide of claim 37.

42. A cDNA according to claim 39 comprising the DNA of SEQ
ID NO: I.

43. A biologically functional DNA vector comprising a DNA
according to claim 37.

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44. A vector according to claim 43 wherein said DNA is
operatively linked to a DNA expression control sequence.

.45. A host cell stably transformed or transfected with a DNA
according to claim 36 in a manner allowing expression of said DNA.

46. A method for producing a macqaque 88C polypeptide
comprising the steps of growing a host cell according to claim 45 in a
suitable nutrient medium and isolating said polypeptide from said cell or
medium.

47. A polynucleotide encoding an 88C polypeptide wherein said
polynucleotide hybridizes under stringent hybridization conditions to the
polynucleotide of SEQ ID NO: 19.

48. A purified and isolated polypeptide comprising the macqaque
chemokine receptor 88C amino acid sequence set out in SEQ ID NO:20.

49. An antibody product that specifically binds a polypeptide
comprising the 88C amino acid sequence set out in SEQ ID NO:20.

50. A hybridoma producing an antibody product according to
claim 49. .

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Full text of "USPTO Patents Application 09870932"

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